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56	Garcı´a-Campan˜a et al.

counting mode, the number of anode pulses per unit time or per unit event is counted. Both photon-counting and current-measuring instruments have a response that is proportional to the sample light output; however, the first mode reads in ‘‘photons per seconds’’ while the second one reads in light arbitrary units, usually referred to as relative light units. The signal recovery performance offered by photon counting has been shown to be superior to the current-detection method over the entire dynamic range of PMT operation. In photon-counting terms this corresponds to rates of a few photons per second to 108/s, equivalent to 1 pA to 100 A in output current. A recent paper discusses the advantages of photon-counting mode versus current-measuring mode, which can be summarized as follows [5]:

The gain process is noisy and introduces a factor into current-detection measurements.

The dark current in current detection always exceeds the dark-current equivalent of dark counts in photon counting because of the leakage. Also, no component of the dark current can be eliminated by discrimination as in photon counting.

It is less sensitive to temperature effects, aging, high voltage stability, rate effects, magnetic effects, and microphonics.

The dynamic range is superior at fixed gains.

The photon-counting method preserves the temporal structure of the signal.

On the other hand, the photon-counting mode has some disadvantages, such as the limited dynamic ranges (while very low light levels can be measured, the maximum measurable light level is relatively low), they are not able to count fast enough to respond to the high light levels and short duration of fast reactions, and they are less sensitive to quenching effects (e.g., methods based on the signal decrease when analyte concentration increases).

Liquid scintillation counters are highly efficient for low CL intensities and consist of two photon-counting channels provided with a variable discriminator. The sample is placed between these two detectors to ensure a high optical efficiency. The discriminator is adjusted to allow photon impacts to be transmitted and small background noise pulses to be rejected. As disadvantages they suffer from saturation errors and provide nonlinear relationships between the CL intensity and the total counts.

Other detection modes in bright CL or BL reactions are multichannel detectors, which provide simultaneous detection of the dispersed radiation and produce a permanent image of a wide area. Photographic films or plates are emulsions that contain silver halide crystals in which incident photons produce stable clusters of silver atoms within the crystals. Internal amplification is provided in the development process by an electron donor that reduces the remaining silver ions to silver atoms within the exposed crystals. A complexing agent is used to remove the

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unexposed silver ions. The photographic detection is an integrating process in that the output (density of silver) is a result of the cumulative effect of the entire incident radiation during the exposing time. The image is constituted by many thousands of grains on each square centimeter of film, only 10–100 photons being necessary to produce a developable grain. The main limitations are: nonlinearity of the response over the wide range of light intensities required to observe CL, restricted dynamic range reciprocity failure, and adjacent effects. More efficient imaging devices for CL are vidicon cameras and charged coupled devices (CCDs), which have recently been incorporated in CL instrumentation. These kinds of detectors are extensively discussed in Chapter 4 and applications of CL imaging are included in Chapter 16.

3.4 Signal Conditioning, Manipulation, and Readout

The photoanodic current from the PMT is first converted to voltage with an operational amplifier in the current-to-voltage configuration. Often, after further voltage amplification the readout signal from the analog transducers is converted to digital because the digital domain allows the signal to be treated with highly accurate digital methods or with software in a microcomputer. Only in the case of the photon-counting mode, the PMT output is directly digital because the registered quantity is the total number of accumulated counts over some boundary conditions. The increasing use of computer-controlled CL instruments allows easy keyboard control of instrumental parameters, and the analytical response is stored in memory for later manipulation. Even specific software has been designed for specialized applications such as kinetics studies and measurements, flow systems CL studies, CL detectors coupled to HPLC or GC, etc.

4. MAIN CHEMILUMINESCENCE APPLICATIONS

In the last few years, interest in the use of CL systems in analytical chemistry has been growing exponentially, mainly in gas and liquid phases; however, CL applications in the solid phase are more limited. Table 2 shows some of the applications of the more widely used CL systems that will be considered in detail in this volume.

Because weak CL emission often is produced from the oxidation of many solid organic compounds, the measurement of this light emission may be used as an indicator of changes in materials composition due to oxidation processes, and for evaluating stabilizers intended to prevent or retard these oxidative alterations [6]. Some examples of materials than can be characterized by CL emission are the polymers that are degraded by weathering, exposure to heat, or exposure to ionizing radiation, or food components that suffer flavor alterations. In this

58		Garcı´a-Campan˜a et al.
Table 2 Common Applications of the Main CL Systems
Phase	Reagent	Analyte
Gas	Ethylene	O3
Gas	O3	Hydrocarbons
		NO
Gas	O3 after conversion of the	Nitrosamines
	analyte to NO	Total nitrogen
Gas	H2 flame	Sulfur compounds
Gas	H2 flame followed by O3	Sulfur compounds
Liquid	Luminol and derivatives	Metal ions and complexes (Co(II), Cu(II),
		Fe(III), Zn(II), Cd(II), Mn(II), Cr(III),
		Cr(IV), Pt(IV), ClO , Fe(CN)63 )
		Heme compounds
		Peroxidases
		Oxidants (H2 O2, O2, I2, etc.)
		Inhibitors (Ag(I), Ce(IV), Ti(IV), V(V), etc.)
		Substances easily oxidized and indirectly de-
		termined (ascorbic, acid, carboxylic acids,
		amines, etc.)
		Substances converted into H2 O2 (glucose,
		etc.)
		Substances labeled with luminol and deriva-
		tives
Liquid	Acridinium esters	Substances labeled with acridinium esters
		Ions (Ag(I), Bi(III), Pb(II), Co(II), Cr(III),
		Cu(II), Fe(III), Mn(II), etc.)
		Oxidants (H2 O2, O2, etc.)
		Substances converted into H2 O2
		Reducing compounds (Cr(II), Fe(II), Mo(V),
		ascorbic acid, tetracyclines, sugars, etc.)
Liquid Ru(bpy)33		Aliphatic amines
Liquid	Peroxyoxalates	Oxidants (H2 O2, etc.)
		Fluorophores (polycyclic aromatic hydrocar-
		bons, etc.)
		Derivatized compounds with fluorophores
		(amino acids, steroids, aliphatic amines,
		carboxylic acids, catecholamines, etc.)
Liquid	Direct oxidation with MnO4 ,	Different molecules (usually in pharmaceuti-
	ClO , Ce(IV), IO4 , etc.	cal applications)
Solid	O2 plus heat	Polymers

Principles, Instrumentation, Applications

59

sense, polyolefins, polyamides, rubber, epoxies, and lubricating or edible oils can be characterized in this way. The methodology used in these cases is based on monitoring of the CL intensity-versus-time curve when the solid is submitted to a controlled high temperature in front of the detector. The shape of this curve is characteristic for the material and can help to identify its composition and characterize its degradation process.

In the past, general chapters and reviews have been published, related to the characteristics of CL as analytical technique [7–9], mainly in the liquid phase [10–14], and its use as detection mode in flowing streams and immunoassay [15– 17]. Two extensive reviews reported on the specific application of CL reactions according to the nature of the analyte (inorganic species, enzymes and nucleotides, acids and amines, carbohydrates, steroids, polycyclic aromatic compounds, and drugs) and covering the literature from 1983 to 1991 [18] and from 1991 to mid-1995 [19].

Specific reviews, books, and chapters reported the applications of BL [20] and CL as a highly sensitive method of detection in the flame [21], in FIA [22, 23], in LC [22, 24, 25], in GC [26], in CE [27–30], and in immunoassays [31, 32]. Application of the most commonly used CL system for postcolumn detection in conventional and in microcolumn LC, the peroxyoxalate-CL reaction, has been revised [33] and the mechanism of this reaction has been critically reviewed and reevaluated [34].

Advances have been achieved in recent years, such as the use of CL reagents as labels to derivatize and sensitively determine analytes containing amine, carboxyl, hydroxy, thiol, and other functional groups and their application in HPLC and CE [35, 36], the synthesis and application of new acridinium esters [37], the development of enhanced CL detection of horseradish peroxidase (HRP) labels [38], the use of immobilization techniques for developing CL-based sensors [39–42], some developments of luminol-based CL in relation to its application to time-resolved or solid-surface analysis [43], and the analytical application of electrogenerated CL (ECL) [44–47], among others.

The number of reactions producing CL as cited in the literature of the last decade is high. Extensive reviews have been reported on analytical applications in different disciplines such as medical and biochemical [48–52], food [53, 54], environmental, and toxicological [55] analysis.

In the gas and liquid phases, very well-established CL reactions exist that have been chronologically introduced in Chapter 1, together with their mechanisms; they will be treated in different chapters of this book. Particularly, some chapters include descriptions of the CL systems and applications in the liquid phase in organic and inorganic analysis (Chapters 5 and 6, respectively), for BL systems (Chapter 10); applications derived from the use of organized media (Chapter 11); the specific study of the mechanism and applications of a widely applied CL system based on the reaction of peroxyoxalates (Chapter 7); kinetics

60	Garcı´a-Campan˜a et al.

considerations in CL analysis (Chapter 8); and the recent applications of ECL (Chapter 9).

As far as the gas phase is concerned, the CL systems that occur at room temperature or in the presence of a flame are extensively described in Chapter 13, together with the applications of these reactions in gas chromatography (GC).

Also, specific chapters deal with the use of CL reactions as detection mode in FIA (Chapter 12), in separational techniques, such as liquid chromatography (LC) (Chapter 14) or capillary electrophoresis (CE) (Chapter 15), in immunoassay (Chapter 18), and in the development of sensors (Chapter 20). The recent use of this technique for the analysis of DNA (Chapter 19) and a photosensitized CL mode for medical routine and industrial applications (Chapter 17) are also considered in this book.

It is clear that the areas of CL and BL, from both the analytical and biochemical/biological points of view, have been the subject of various international series of symposia that have been organized since the early 1970s, and the importance of which apparently is increasing for the next decade, as can be noticed from the calendar of events published by reputed international journals. An exciting analytical future is hence to be expected.

5.CHEMILUMINESCENCE AND BIOLUMINESCENCE ON THE INTERNET

Recently Stroebel et al. published the first survey of Internet sites relating to various aspects of luminescence [56]. In their classification they cite relevant companies and existing products, general websites, images, movies, and demonstrations for educational purposes. Below are summarized some of these useful websites to which others have been added.

5.1 General Websites

Analytical Chemiluminescence. Naval Air Warfare Center Weapons Division NA WCWPNS, China Lake. Chemiluminescence history.

http://www.nawcwpns.navy.mil/clmf/chemilume.html Chemiluminescence.

http://fischer.union.edu/chem20/experiments/chemiluminescence.htlm Chemiluminescence.

http://www.deakin.edu.au/ swlewis/cl.htm

Chemiluminescence and laser-induced fluorescence spectra of lightsticks. http://www.cs.moravian.edu/chemistry/lightstick/1_st_scheme.html

Chemiluminescence definitions and primer.

http://www.shsu.edu/ chm_tgc/chemilumdir/Define.html

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Chemiluminescence detector.

http://www.techlab.de/produkte/c1-2.html Chemiluminescence-light without heat.

http://scifun.chem.wisc.edu/homeexpts/chemilum.html Chemiluminescence of tris ruthenium(ll) ion.

http://chemed.chem.purdue.edu/ genchem/demosheets/5.12.html Chemiluminescence reaction.

http://learn.chem.vt.edu/user/long/demo/luminol.html Chemiluminescence sites of interest.

http://www.shsu.edu/ chm_tgc/chemilumdir/chemisites.html Chemiluminescence spectroscopy.

http://www.scimedia.com/chem-ed/spec/molec/chemilum.htm Luminol/hydrogen peroxide reaction.

http://www.pky.ufl.edu/homepages/faculty/eg/demo.html

Bioluminescence production of light by living organisms resulting from the conversion of chemical energy to light energy.

http://www.encyclopedia.com/printable/01486-a.html The Bioluminescence Web Page.

http://lifesci.ucsb.edu/ biolum/index.shtml

5.2 Images, Movies, and Demonstrations

Chemiluminescence movie.

http://www.shsu.edu/ chm-tgc/chemilumdir/movie.html Chemiluminescence: firefly reaction Quick-Time video.

http://www.newlisbon.k12.wi.us/reactions/home.html%234 Chemiluminescence: glowing tornados.

http:1/learn.chem.vt.edu/user/long/demo/chemiluminescence.html Chemiluminescence: the Cyalume lightstick (demonstration).

http://chemed.chem.prudue.edu/ genchem/demosheets/5.8.html The Chemiluminescence Home Page.

http://www.shsu.edu/ chm_tqc/chemilumdir/chemiluminescence2.html Demonstration of chemiluminescence. Luminol chemical concept.

http://chemed.chem.purdue.edu/ genchem/demosheets/5.9.html Chemiluminescence experiments.

http://library.advanced.org/3310/nographics/experiments/lumine.html

5.3 Companies, Instruments, and Products

Andor technology

http://www.andor-tech.com/

62 Garcı´a-Campan˜a et al.

Argus Laboratories Ltd. Chemiluminescent and bioluminescent products Berthold. Luminescence assays

http://www.berthold-online.com/

http://www.net-escape.co.uk/business/argus/

Biosynth. Bioluminescent products list, bioluminescent substrates, and related products.

http://www.biosynth.inter.net/page14.html Chemiluminescence

http://www.etd.ameslab.gov/etd/technologies/projectss/chemilum.html Hamamatsu. Photomultipliers and photon countings for bioluminescence and

chemiluminescence. http://www.hamamatsu.com/

Kodak. Chemiluminescence imaging http://www.nenlifesci.com/

Lumigen, Inc.

http://www.lumigen.com/

McPherson, Inc. Model 660 HPLC Chemiluminescence detector.

http://www.mcphersoninc.com/hplcdetectors/mode1660description.htm Ocean Optics. Optical accessories

http://www. OceanOptics.com/

Oriel. Photomultipliers and electronic and optical devices http://www.oriel.com/

Photometrics. High-performance CCD imaging for bioluminescence. http://www.photomet.com/images/im.biolum.html

Photomultipliers for bioluminescence and chemiluminescence. http://www.electron-tubes.co.uk/pmts/pmtchem.html

Photosensitized chemiluminescence http://www.riat.com/

Photoreactors

http://www.bioanalytical.com/

Sievers Instruments. Gas-phase chemiluminescence detector http://www.SieverInst.com/

Tropix, Inc.

http://www.tropix.com/

Turner Designs TD-20/20 luminometer.

http://www.seoulin.co.kr/protocol/turner/chem.htm

It is clear that when surfing on the Internet applying uniquely the keyword ‘‘chemiluminescence,’’ many important sites show up. These include principles and applications, spectroscopy, potentials in liquid chromatography, university training courses, products, demonstrations, etc.

The journal Luminescence (The Journal of Biological and Chemical Luminescence) includes a section featuring new products, people, and conferences,

Principles, Instrumentation, Applications

63

regular literature reviews, and abstracts from Conference Proceedings (http:// www.interscience.wiley.com/jpages/0884-3996/). Interesting also is the website of the International Society for Bioluminescence and Chemiluminescence (http:// www.unibo.it/isbc/).

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