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Hindawi Publishing CorporationISRN SpectroscopyVolume 2013 Article ID 230858 12 pageshttpdxdoiorg1011552013230858

Review ArticleA Recent Review on Chemiluminescence Reaction Principle andApplication on Pharmaceutical Analysis

Tadesse Haile Fereja1 Ariaya Hymete2 and Thirumurugan Gunasekaran1

1 Department of Pharmacy College of Medicine and Health Sciences Ambo University Ambo 19 Ethiopia2Department of Pharmaceutical Chemistry and Pharmacognosy Addis Ababa University Addis Ababa 117 Ethiopia

Correspondence should be addressed to Tadesse Haile Fereja tdss haileyahoocom

Received 16 July 2013 Accepted 15 September 2013

Academic Editors V M Bermudez H J Byrne and Z Wang

Copyright copy 2013 Tadesse Haile Fereja et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

This paper provides a general review on principle of chemiluminescent reactions and their recent applications in drug analysisThestructural requirements for chemiluminescent reactions and the different factors that affect the efficiency of analysis are includedin the review Chemiluminescence application in immunoassay is the new version for this review Practical considerations are notincluded in the review since the main interest is to state through the aforementioned applications that chemiluminescence hasbeen is and will be a versatile tool for pharmaceutical analysis in future years

1 Introduction

Luminescence is the most conveniently defined as the radi-ation emitted by a molecule or an atom when these speciesreturn to the ground state from the exited state According tothe source of excitation luminescence phenomenon could beclassified as photoluminescence (fluorescence and phospho-rescence) when the excitation source is energy from absorbedlight chemiluminescence-energy from chemical reactionsand bioluminescence energy frombiologically catalysed reac-tions An exitedmolecule has the same geometry and is in thesame environment as it was in ground state In this situation iteither emits a photon from the same vibrational level towhichit was exited initially or it undergoes in vibrational level priorto emission of radiation [1 2] For an isolated molecule in thegas phase the only way to lose vibrational energy is to emitan infrared photonwhich is less probable thanundergoing anelectronic transition to return to the ground state Thereforeone tends to see photon emission from higher vibrationallevels of exited states in gas phase spectra at low pressure In asolution however thermal relaxation of a vibrationally exitedmolecule is quite rapid through transfer of excess vibrationalenergy from the solute molecule to the solvent In fact thisprocess is so efficient that all the excess vibrational energy

of the exited state is lost and this process occurs in 10minus13to 10minus11 sec This means that before an exited molecule ina solution can emit a photon it will undergo vibrationalrelaxation and therefore photon emission will always occurfrom the lowest vibrational level of an excited state [3 4]

Once a molecule arrives at the lowest vibrational level ofan exited singlet state it can do a number of things one ofwhich is to return to the ground state by photon emissionThis process is called fluorescence The lifetime pf an exitedsinglet state is approximately 10minus9 to 10minus7 sec and therefore thedecay time of fluorescence is of the same order of magnitudeThe quantum efficiency of fluorescence is defined as thefraction of molecules that will fluoresce

In addition to fluorescence one also encounters radia-tionless process where molecules in an excited singlet statemay return to the ground state without the emission of aphoton converting all the excitation energy into heat theprocess called internal conversion Generally internal con-version is an inefficient process and is probably only a smallfraction of the total excitation energy in most moleculesAlthough population of triplet states by direct absorptionfrom the ground state is insignificant a more efficient processexists for population of triplet states from the lowest excitedsinglet state in many molecules This process is referred

2 ISRN Spectroscopy

Singlet excited states

Internalconversion

Vibrationalrelaxation

Ener

gy

Groundstate

Absorption Fluorescence

Triplet excited state

Intersystemcrossing

Internaland

externalconversion

Phosphorescence

T1

1205822 12058211205823120582r 1205824

120582998400

r

S1

S2

S0Vibrationalrelaxation

Figure 1 Jablonski diagram describing the electronic levels of common organic molecules and possible transitions between different singletand triplet states (M Sauer J Hofkens and J Enderlein Handbook of Fluorescence Spectroscopy and Imaging)

to as intersystem crossing and is a spin-dependent internalconversion process A molecule in excited triplet state maynot always use intersystem crossing to return to the groundstate It could loss energy by emission of a photon the processcalled phosphorescence A triplet-singlet transition is muchless probable than a singlet-singlet transition The lifetime ofthe excited triplet state can be up to 10 sec in comparisonwith 10minus5 s to 10minus9 s of average lifetime of an excited singletstate Emission from triplet-singlet transition can continueafter initial irradiation [19 20]

2 The Nature of ChemiluminescenceReactions

In general chemiluminescence reaction yields one of thereaction products in an electronic excited state producinglight on falling to the ground state As can be seen in Figure 1the process of light emission in chemiluminescence is thesame as in photoluminescence except for the excitation pro-cess In fluorescence and phosphorescence the electronicallyexcited state is produced by absorption of Uv-visible lightreturning to the ground state (S

0

) from the lowest singletexcited state (S

1

) or from the triplet excited state (T1

) [21 22]In general a chemiluminescent reaction can be generated

by two basic mechanisms (Figure 2) in a direct reactiontwo reagents usually a substrate and an oxidant in thepresence of some cofactors react to from a product orintermediate sometimes in the presence of a catalyst Thensome fraction of the product or intermediate will be formedin an electronically excited state which can subsequentlyrelax to the ground state with emission of a photon Thesubstrate is the CL precursors which is converted into the

electronically excitedmolecule responsible for light emissionor acting as the energy as transfer donor in indirect CL Thecatalyst enzyme or metal ions reduce the activation energyand provide an adequate environment for producing highCL efficiency out of the process Cofactors sometimes arenecessary to convert one ormore of the substrates into a formcapable of reacting and interacting with the catalyst or toprovide an efficient leaving group if bond cleavage is requiredto produce the excited emitter On the contrary indirect orsensitized CL is based on a process of transfer of energy ofthe excited speciersquos to a fluorophore [23] This process makesit possible for those molecules that are unable to be directlyinvolved in CL reaction to transfer their excess of energy toa fluorophore that in turn is excited releasing to its groundstate with photon emission All of these paths lead to a greatvariety of practical uses of CL in solid gas and liquid phases[24]

21 Requirements for Chemiluminescence Emission For achemical reaction to produce light it should meet someessential requirements

(1) The reactionmust be exothermic to produce sufficientenergy to form the electronically excited state Topredict whether the CL reaction will occur or not itis possible to use free energy (Δ119866)

Δ119866 = Δ119867 minus 119879Δ119878 (1)

where 119879 is temperature Δ119867 is enthalpy and Δ119878 isentropy

ISRN Spectroscopy 3

A + B +(Chemiluminescent (Oxidant)

(Cofactors)

Directchemiluminescence

Catalyst

Senitized or energy transferchemiluminescenceF

precursor)

F + h

P + h

P + h

Plowast (intermediate or product)

Figure 2 Types of CL reaction P product F fluorescing substance

Enthalpy is the actual energy source for generatingthe electronically excited state product To initiatethe chemical reaction the activation energy (Δ119867

119860

)is absorbed The available energy to produce theexcited state (Δ119864EX) must be the difference betweenthe reaction energy and the activation energy If thedifference is equal to or greater than the energyrequired for generating the excited state Δ119864EX the CLprocess will be produced Consider

Energy Available = Δ119867119860

minus Δ119867119864

ge Δ119864EX (2)

In many CL reactions the entropy change is small soΔ119866 and Δ119867 are very similar in magnitude and theenergetic requirements can be established in termsof (Kcalsdotmolminus1) In this sense for CL to occur thereaction must be sufficiently exothermic such that

Δ119866 geℎ119888

120582ex=286 times 10

4

120582ex (3)

where 120582ex is the long wavelength limit (nanometers)for excitation of the luminescent species Becausemost of the CL reaction produce photons in therange 400 (violet)ndash750 (red) nm the creation of theelectronically excited state and the generation ofCL inthe visible region require around 40ndash70Kcalsdotmolminus1This exothermic condition is associated with redoxreactions using oxygen and hydrogen peroxide orsimilar potential oxidants [25]

(2) The reaction pathway must be favorable to channelthe energy for the formation of an electronicallyexcited state In case the chemical energy is lost asheat for example via vibration and rotational energyways the reaction will not be chemiluminescent

AB + D

D

BA

(6)

(5)

(7)

ABlowast

lowastA

A + B

BF +

ACC

+ B

AB + F

(3)

(2)

(4)

AB + light

Figure 3 different processes for losing energy from the excited state(1) direct CL (2) molecules dissociation (3) chemical reaction withother species (4) intramolecular energy transfer (5) intermolecularenergy transfer (in case of a fluorphore indirect CL) (6) isomeriza-tion and (7) physical quenching

(3) Photon emission must be a favorable deactivationprocess of the excited product in relation to othercompetitive nonradiative processes that may appearin low proportion (Figure 3) in the case of sensitizedCL both the efficiency of energy transfer from theexcited species to the fluorophore and the fluroesenceefficiency of the latter must be important

In all the luminescent process the intensity of theproduced emission depends on the efficiency of generatingmolecules in the excited state which is represented by thequantum efficiency (quantum yield) and the rate of thereaction In the case of CL reactions the intensity can beexpressed as

119868CL = OslashCL minusd119860d119905 (4)

where 119868CL is the CL emission intensity (photonssecond) OslashCLis the CL quantum yield and (minusd119860d119905) is the rate at whichthe CL precursor 119860 is consumed Higher values of quantumyields are usually associated with BL reactions whereas inmost of the CL reactions used for analytical purposes rangefrom 0001 to 01 and even very inefficient systems with muchlower quantum yields can be used in analysis based on thecomplete absence of background emission This is the casewith ultraweak CL reactions in which can be less than 0001(often 10minus3ndash10minus2) and sometimes 10minus9ndash10minus15

Ultra-weak CL is produced from oxidative reaction inliving cells and the emitted signals are mostly 103ndash106 timesless intense than those from luminous organisms The maincharacteristic of ultraweak CL emission is that it is invisibleto the naked eye This kind of CL includes a group of CLreactions that at least in living cells involve a number ofoxygen intermediate that play an important role in certaintypes of cell activation in the defense system of the body andin ischemic heart diseases It has been detected from a widevariety of infact organs isolated cells and tissue homogenatesfrom vertebrates invertebrates plants and from several

4 ISRN Spectroscopy

reactions in vitro Ultra-weak CL is associated with someimportant cellular functions such as mitochondrial respira-tions photosynthesis cell division or phagocytosis amongothers [26ndash28]

22 Some Factors Influencing Chemiluminescencersquos EmissionBecause of the cited dependence of CL intensity uponvarious parameter CL measurementrsquos are strongly modifiedby experimental factors that affect quantum yield and rate ofreaction such as

(i) the chemical structure of the CL precursor notonly the central portion containing the electronicallyexcited group but also the side chain

(ii) the nature and concentration of other substratesaffecting the CL pathway and favoring other nonra-diative competition process

(iii) the hydrophobicity of the solvent and solution com-position as example the of luminal oxidized indimethylusfoxide (DMSO) is 005 comparedwith 001in water the colors being blue-violet (425 nm) andblue green (480ndash502 nm) respectively

(iv) the presence of energy transfer acceptors [29]

221 Chemical Considerations A sine qua non for CL tooccur is that the precursor(s) of the light-emitting speciesmust participate in reaction that releases a considerableamount of energy For visible emission (say 400ndash750 nmin wavelength) 40ndash70 kcalsdotmol is required Actually theenthalpies of reaction have to be slightly greater than thesevalues owing to energy losses by thermal relaxation in theground state (after emission) and lowest excited singlet (afterexcitation) states of the fluorophore Normally only certainoxidation-reduction reactions generate this much energyIn addition at least some of the energy produced mustbe channeled into a reactions pathway in which at leastone of the upper vibration levels of the reactants probablycorresponding to the transition state of the initial reactionhas the same energy and a comparable geometrical structureas an upper vibrational level of the lowest excited singletstate of potentially emissive product of the reaction Thegeometric identity or near-identity of the interconvertingspecies ensures that most of the energy of activation ischanneled into free energy of activation rather than beingwasted as entropy or activation [30 31]

Invariably there will also be pathway for dark reactionThe dark reaction may lead directly to the ground electronicstate species that also results from the fluorescence of theexcited product but this is not necessarily so It is possible tohave a competing dark reaction that leads directly to groundstate products different from that produced by fluorescenceIf Δ119867

4

is the sum of the enthalpies of activation for all darkreaction competing with the chemilluminercent pathwaywhose enthalpy of activation is Δ119867

4119894

CL will be probablewhen Δ119867

4

lt Δ1198674119894

(38) This occurs when the lowest excitedsinglet state of the flurophore has the same geometricalconfiguration as the ground electronic state of the reactants

Pote

ntia

l ene

rgy l

evel

Distance between nuclei(r)

ΔH

ΔH

Figure 4 Relative positions of the potential energy (119864) surface ofthe electronic states involved in a hypothetical chemiluminescentreaction as a function of internuclear separation (119903)

at lower energy than when the dark product in the groundelectronic state hase the same configuration as the reactantsin their ground electronics stateThis is illustrated in Figure 4Often catalysts which are usually transition metal ions inchemilluminercent reaction and enzymes in bioluminescentreactions are employed to make light emitting reactionsprocessed at a convenient rate This is accomplished by thecatalyst forming a transient complex with reagents WhoseΔ1198674119894

is lower than that of unanalyzed reaction The catalystis usually a cooxidant that is it assists electron transferThe catalysis of the luminol hydrogen peroxide reaction byCo(II) and of the luciferian-oxygen reaction by the enzymeluciferase are important examples (613)119901 and 119901lowast represent the ground and lowest electronically

excited singlet states of the product of the reaction respec-tively and 119877 represents the ground electronic state of thereactant Δ119867 is the enthalpy of the ldquodarkrdquo reaction whileΔ1198674

is the enthalpy of activation Δ1198674

is the enthalpy ofactivation of the photoreaction hv that denotes the emissionof chemiluminescence [32]

222 Photophysical Considerations Subsequent to the for-mation of the potentially CL molecule in its lowest excitedsinglet state a series of events carry the excited moleculedown to its ground electronic state Since the electronicallyexcited molecule is initially also vibrationally excited rapidstepwise (10minus13ndash10minus12 s) thermal deactivation of the excitedmolecule called thermal or vibrational relaxation in whichthe molecule loses vibrational energy by inelastic collisionswith the solvent occurs The process of vibrational relaxationcarries the molecule to the lowest vibrational level of thelowest excited singlet state Certain molecules may returnradiationlesly all the way to the ground electronic state in thesame time frame by thermal deactivation a process known asinternal conversion However in some molecules for a vari-ety of reactions the return from the lowest vibrational level ofthe lowest excited singlet state to the lowest vibrational levelof the ground state by internal conversation and vibrationalrelaxation is forbidden (ie of low probability or longduration) In thesemolecules return to the ground electronic

ISRN Spectroscopy 5

state occurs by one of two alternative pathways the simplerbeing the direct emission of ultraviolet or visible radiationwhose frequency or wavelength is governed by the energygap between the lowest excited single state and the groundelectronic state [33] The radiative transition between excitedand ground state of the same spinmultiplicity occurs in a timeframe 10minus11ndash10minus7 s after excitation and is called fluorescenceOwing to the fact that the ground electronic state of moleculehas several vibrational levels associated with it fluorescenceemission does not occur at a singlewavelength but rather overa range of wavelength corresponding to several vibrationaltransitions as components of a single electronic transition[34]

Several processes may compete with fluorescence fordeactivation of the lowest excited singlet state As a resultonly a fraction of the molecules formed in the lowest excitedsinglet state Oslash Actually fluoresce Oslash is called the quantumyield or fluorescence efficiency It is usually a fraction butmay be unity in some exceptional cases and is related tothe probabilities (rate constant) of fluorescence (119896ef) andcompetitive process (119896

119889

) [35]

223 Structural and Environmental ConsiderationMolecules that enter into CL reaction are generally reducedspecies that can be easily oxidized Molecules containingamino and hydroxyl groups fall into this category as dopolycyclic aromatic ring systems Under strongly alkalineconditions hydroxyl groups and even some arylamino orarylamido groups can be deprotonated making them evenmore susceptible to oxidation (Table 1) On the other handelectron-withdrawing groups tend to stabilize electroniccharge and make oxidation more difficult in moleculesto which they are affixed Form the chemical point ofview the solvent in which the CL experiment is carriedout can have a dramatic influence on the efficiency of theCL reaction as salvation can alter the shapes the depthsand the densities of the vibrational states of the potentialsurfaces representing the ground states of products andreactants and the lowest excited singlet state of the potentialfluorophore The alteration of the intersections of thesepotential energy surface an affect the enthalpies of reactionand the enthalpies of activation for dark and lumigenicreactions In some cases these changes will favor CL (if Δ119867lowastdecreases relative to Δ119867lowast) and in some cases they will makeit thermodynamically unfavorable for CL to occur Otherinfluences of the structure and the environment are manifestin the rates of processes competing with fluorescence fordeactivation of the lowest excited singlet state These arethe process and properties that influence the fluorescenceprocess [36 37]

224 The Influence of Molecular Structure on FluorescenceAmong other factors the quantum yield of fluorescencedetermines the intensity of lights emission in a CL This aswell as the position in the spectrum occupied by the fluores-cence band is largely a function of the molecular structureFluorescence is most often observed in highly conjugated oraromatic organic molecules with rigid molecular skeletons

The less vibrational and rotational freedom that themoleculeshas the greater is the probability that the energy gap betweenthe lowest excited singlet state and the ground electronicstate will be large so that fluorescence will predominateover nonradiative deactivation and assure a high fluorescenceefficiency

Aromatic molecules containing freely rotating sub-stituents usually tend to fluoresce less intensely than thosewithout these substituentsThese result from the introductioninto each electronics state of rotational and vibrationalsubstates by the exocyclic substituents [37]

225 The Influence of the Environment onthe Fluorescence Spectrum

Solvent Effect Solvent interactions with molecules are mainlyelectronic and it is usually the difference between theelectrostatic stabilization energies of ground and excitedstate that contribute to the relative intensities and spectralpositions of fluorescence in different solvents The change inelectron distribution that takes place upon transition fromthe lowest excited singlet state to the ground state duringfluorescence causes changes in the dipolar and hydrogen-bonding properties of the solute If the solute is more polarin the excited state than in the ground state fluorescencewill occur at longer wavelength in a polar solvent than ina nonpolar solvent This is because the more polar solventwill stabilize the excited state relative to the ground stateMoreover the fact that photoluminescence originates froman excited state that is in equilibriumwith its solvent cage andterminates in a ground state that is not cause the fluorescenceto lie at longer wavelengths the more polar or more stronglyhydrogen-bonding the solvent [38]

The lowest excited singlet state is formed by promoting alone-pair electron to a vacant 120587 orbital (this is called an 119899 120587lowaststate) These molecules tend to show very little fluorescencein aprotic solvents such as aliphatic hydrocarbons becausethe 119899 120587lowast excited singlet state is efficiently deactivated byintersystem crossing However in portic solvents such aswater to ethanol these molecules become fluorescent Thisresults from the destabilization of the lowest singlet 119899120587lowast stateby hydrogen bonding If this interaction is sufficiently strongthe fluorescent120587120587lowast state drops below the 119899120587lowast state allowingintense fluorescence Quinoline for example fluoresces inwater but not in cyclohexane [39]

Solvents containing atoms of high atomic number (egalky iodide or bromides) also have a substantial effect on theintensity of fluorescence of solute molecules Atoms of highatomic number in the solvent cage of the solute moleculeenhance spin-orbital coupling in the lowest excited singletstate of the solute This favors the radiationless populationof the lowest triplet state at the expense of the lowest excitedsinglet stateThus in heavy-atom solvents all the other thingsbeing equal fluorescence is always less intense than that insolvent of low molecular weight [40]Quenching of Fluorescence Fluorescence may be decreasedor completely eliminated by interactions with other chemical

6 ISRN Spectroscopy

Table 1 Common application of the main CL System

Phase Reagents Analyte ReferencesGas Ethylene O3 [5]

Gas O3 after conversion of the analyteto NO Nitrosamine total nitrogen [6]

Gas H2 flame Sulfer compounds [7]Gas H2 flame followed by O3 Sulfer compounds [7]

Liquid Luminal and derivatives

Metal ions and complexes (Co(II) Cu(II) Fe(III) Zn(I) Cd(II)Mn(II) Cr(III) Cr(Iv) Pt(Iv) ClOminusFe(CN)

6

minus3Heme compounds Peroxides oxidants (H2O2 O2 I2 etc)Inhibitors (Ag(I) Ce(IV) Ti(IV)Substance easily oxidized and indirectly determined (Ascorbic acidcarboxylic acids amines etc )Substances converted in to H2O2 (glucose etc )Substances labeled with luminal and derivatives

[8ndash13]

Liquid Acridinium esters

Substance labelled with acridinium esters Ions Ag(I) Bi(III) Pb(II)Co(II) Cr(III) Cu(II) Fe(III) Mn((II) etc)Oxidants (H2O2 O3 etc ) Substances converted in to H2O2Reducing compounds (Cr(II) Fe(II) Mo(V) ascorbic acidstetracyclines sugars etc )

[14 15]

Liquid Peroxyoxalates

Oxidants (H2O2 O3 etc ) Flourophores (polycyclic aromatichydrocarbons etc ) Derivativatized compounds with fluorophores(amino acids steroids aliphatic amines carboxylic acidscatecholamines etc )

[10 12]

Liquid Direct oxidation with MnO4ClOminus Ce(Iv) IO

4

minus etc Different molecules (usually in pharmaceutical applications) [16ndash18]

species This phenomenon is called quenching of fluores-cence Obviously if the fluorescence of fluorophore generatedin CL reaction is quenched the observation of chemilumines-cence will be precluded

Two kinds of quenching are distinguished In staticquenching interaction between the potentially fluorescentmolecule and the quencher takes place in the ground stateforming a nonfluorescent complexThe efficiency of quench-ing is governed by the formation constant of the complex aswell as by the concentration of the quencher The quenchingof the fluorescence of salicylic acid by Cu(II) is an example

In dynamic quenching (or diffusional quenching) thequenching species and the potentially fluorescent moleculereact during the lifetime of the excited state of the latter Theefficiency of dynamic quenching depends upon the viscosityof the solution the lifetime of the excited state (pound

119902

) of theof the luminescent species and the concentration of thequencher [119876] This is summarized in the Stern-Volumerequation

OslashOslash119902

=1

1 + 119896119876

(pound119902

) [119876]

(5)

where 119896119876

is the rate constant for encounter between quencherand potentially luminescing spices and Oslash and Oslash

119902

are thequantum yields of fluorescence in the absence and presenceof concentration [119876] of the quencher respectively119896119876

Is typical of diffusion-controlled reactions(sim1010Mminus1 sminus1) white for a fluorescent molecule is typically10minus8 or less Hence 119896

119902

le 102Mminus1 and for dynamic quenching

to be observed (say1 quenching) [119876] must be greater thanor equal to 10minus4M [41 42]

Energy Transfer Energy transfer entails the excitation of amolecule that during the lifetime of the excited state passesits excitation energy to othermoleculesThe loss of excitationenergy from the initial excited species (the door) results inquenching of the luminescence of the energy donor and mayresult in luminescence from the energy recipient (acceptor)which becomes excited in the process

Energy transfer can occur by either or two acceptor con-centration dependent processes In the resonance excitationtransfer mechanism or dipole mechanism the donor andacceptor molecules are not in contact with one another andmay be separated by as much as 10 nm (although transferdistance closer to 1 nm are more common) In the classicalsense the excited energy donor molecule may be thoughtof as transmitting antenna that creates an electrical field inits vicinity Potential acceptor molecules within the range ofthis electrical field function as receiving antennae and absorbenergy from the field resulting in their electronic excitationThe rate of resonance energy transfer decreases with the sixthpower of the distance between the donor and acceptor dipolesaccording to

119870ET =1

pound119863

1198770

119877 (6)

where 119870ET is the rate constant for resonance energy transferand pound

119863

is the lifetime of the excited state of the donor

ISRN Spectroscopy 7

molecule 119877 is the mean distance between the center forthe donor and acceptor dipoles and 119877

0

is constant fora given donor-acceptor pair corresponding to the maindistance between the centers of the donor and acceptordipoles for which energy transfer from donor to acceptorand florescence from the donor are equally probable Anothergeneral requirement for the occurrence of resonance energytransfer is the overlap of the fluorescence spectrum of thedonor and the absorption spectrum of the acceptor Anydegree of overlap of these spectra will satisfy the quantizationrequirements for the energy or the thermally equilibrateddonormolecules to promote the acceptor to a vibrational levelof excited singlet state The greater the degree or overlap ofthe luminescence spectrum of the donor and the absorptionspectrum of the acceptor the greater is the probability thatenergy transfer will take place [43 44]

The exchange mechanism of excitation energy transferis important only when the electron clouds of donor andacceptor are in direct contact In this circumstance thehighest energy electrons of the donor and acceptor mayexchange places Thus the optical electron of an exciteddonor molecule may become part of the electronic structureof an acceptor molecule originally in the ground singlet statein while the donor is returned to its ground singlet state byacquiring an electron from the acceptor Exchange energytransfer is also the most efficient when the fluorescencespectrum of the donor overlaps the absorption spectrumof the acceptor Exchange energy transfer is a diffusion-controlled process (ie every collision between donor andacceptor leads to energy transfer) and such its rate depends onthe viscosity of the medium Resonance energy transfer onthe other hand is not diffusion-controlled does not dependupon solvent viscosity and may be observed at the lowestconcentrations of acceptor species Energy transfer from theinitially chemiexcited species to a suitable acceptor flowed byfluorescence from the acceptors in an important process inchemiluminescence [45 46]

23 Chemiluminescence Reaction Parameters CL reaction iscommonly divided into two classes In the type I (direct)reaction the oxidant and reductant interact with rate constant119896119903

to directly from the excited product whose excited singletstate decays with the first (or pseuodfrist)-order rate constant119896119903

= 119896119891

+ 119896119889

In type II (indirect) reaction the oxidant andreactant interact with the formation of an initially excitedproduct (119896) followed by the formation of an excited secondaryproduct either by subsequent chemical reaction or by energytransfer with constant119870The secondary product then decaysfrom the lowest excited singlet state with rate constant 119896 TypeII reactions are generally denoted as a complex or sensitizedchemiluminescence

If OslashCL is the efficiency of the chemiluminescence reac-tion which is the ratio of the number of photons emitted tothe number ofmolecules of reactant reacting it can be definedfor a type I reaction as

OslashCL = Oslash119877

times Oslash119864

times Oslash119891

(7)

where Oslash119877

the chemical yield is the ratio of the numberof molecules that react through the chemilluminercentpathway to the total number of molecules reacted Oslash

119864

theexcitation yield is the ratio the number of molecules thatform an electronically excited product to the number ofmolecules that react through theChemiluminescent pathwayand Oslash

119891

is the quantum yield of fluorescence of the light-emitting species

In type II reaction


times Oslash119864

times Oslash119891

times OslashET (8)

where all symbols have the same meanings as above andOslashET is the efficiency of energy transfer from the initiallychemiexcited species to the energy transfer acceptor OslashCLofcourse a function of the chemical and photo physicalfactors described in Section 222 such as solvent polarityreagent concentrations and molecular structure

The physical significant of OslashCL is the under definedexperimental conditions it is the constant of proportionalitybetween the observe intensity of chemiluminescence andthe rate of consumption of the initial luminophore (reactantL) that is 119897CL = OslashCL times (minusd119871d119905) [47 48]

3 Chemiluminescence Based Analysis

Because the reaction rate is function of the chemical concen-tration CL techniques are suitable for quantitative analysisThe usefulness of CL system in analytical chemistry is basedon some special characteristic

(1) As the technique simultaneously comprises kineticand luminescence features it provides highly sensi-tive and wide dynamic ranges Excellent detectionlimits in the range of femtomoles can be reached ifOslashCL is high enough As an example in the gas phasetypical detection limits are 10 pmol NO using ozoneand 01 pmol for sulfur compounds using a hydrogenflame followed by ozone in the liquid phase downto 1 fmol of the fluorophore may be detected usingthe peroxyoxlalte CL system and 01 fmol peroxidasewhen using luminol In comparison to other spec-trometric techniques in general CL is approximately105 timemore sensitive than absorption spectrometryand at least 103 times more sensitive than fluorimetry

(2) Compared to photoluminescence process no externallight source is required which offers some advantagessuch as the absence of scattering or backgroundphotoluminescence signal the absence of problemsrelated to instability of the external source reductionof interferences due to a nonselective excitation pro-cess and simple instrumentation

(3) Selectivity and linearity are most dependants on thereaction conditions chosen As for photolumines-cence process absorption or emission radiation bythe analyte products or concomitants can causenonlinearity or spectral interference

8 ISRN Spectroscopy

(4) The technique is versatile for determination of awide variety of species that can participate in theCL process such as CL substrates or CL precursorsresponsible for the excited state the necessary reagentfor the CL reaction (usually an oxidant) some speciesthat affects the rate or efficiencies of CL reactionactivators such as catalyst (enzymes or metal ions)or inhibitors such as reductants that inhibit the CLemission fluorphores in the case of sensitized CLsome species that are not directly involved in theCL reaction but that can react with other reagentsin coupled reactions to generates a product that isa reactant in the CL reaction species that can bederivatized with some CL precursors or fluorophoresbeing determined by direct or sensitized CL

(5) Depending on the nature of the analyte and the CLreaction the increase or decrease of CL intensity willbe directly related to the analyte concentration

(6) CL reactions can be coupled as a detection tech-nique in chromatography capillary electrophoresisor immunoassay providing qualitative andor quan-titative information or a large variety of species in thegas and liquid phases

(7) CL reaction can be coupled as a detection tech-nique in chromatography capillary electrophoresis orimmunoassay providing quantitative andor quanti-tative information of a large variety of species in thegas and liquid phase

As an empirical rule CL behavior may be predicated ina compound or its derivatization product has fluorescenceproperties It is possible that oxidization of such a speciesmayproduceCL but there aremany exceptions to this general rule[49ndash51]

31 Instrumentation The instrumentation employed forchemiluminescence measurements basically consists of amixing device and a detection systemThree approaches havebeen used to measure the intensity of emitted light

The first approach involves the use of static measurementsystem in which the mixing of reagents is performed in avessel held in front of the detector The chemiluminescencereagent is added to the analyte in a cuvette held in adark enclusure and the intensity of the light emitted ismeasured through an adjacent photomultiplier tube In thisstatic system the mixing is induced by the force of theinjection This simple reagent addition system without amixing device is of limited usefulness in measuring fastchemiluminescent reactions with precisionThe procedure isalso rather cumbersome requiring separate cuvettes for eachmeasurements as well as repeated opening of the light-tightapparatus which necessitates special precautions to protectthe photomultiplier tube

The second approach is the two-phase measurementsystem In this system the chemilluminercent reagents areimmobilized on a solid support such as filter paper and theanalyte is permitted to interact with the immobilized reagentby diffusion or convection The light emitted is measured

using a micro liter plate reader or by contact printing withphotographic detection The chemilluminercent intensity ofthese systems is influenced by both the kinetics of the reactionand the efficiency of the mass transfer process bringing thereactants together The principal advantages of this systemlie in the conservation of reagents and the convenience ofmeasurement

The third approach involves the use of flowmeasurementsystems The flow injection approach has been described asthe most successful of the methods It involves injection ofthe analyte into a steam of appropriate pH remote fromthe detector and the chemiluminescent reagent flows inanother stream The two streams meet at a T-junction insidea light-tight enclosure then flow through a flat coil placedimmediately in front of a photomultiplier tubeThis compactassembly provides more rapid and reproducible mixingresulting in reproducible emission intensities and permittingrapid sample through put The design of the mixing deviceand themeans of retaining the emitting solution in viewof thedetectors are of important consideration Mixing is reportedas being the most effective at a T-piece or a Y-junctionalthough some have used a conventional FIA inwhich sampleis injected in to the surrounding flowing reagent to achievemixing This approach is reproducible but reported as notproducing a rapid mixing In flow measurement system thechemiluminescence signal has to be measured during themixing as a result only a section as opposed to the wholeintensity-time curve is measured An additional feature ofthese systems is that the shape of the curve now dependson both the kinetics of the chemilluminercent reaction andthe parameters of the flow system Chemical variables suchas reagent concentration and pH and physical parameterssuch as flow rate reaction coil length sample size and thelimitations of experimental apparatus all affects the perfor-mance of flow injection proceduresThe effects of these on theobserved analytical signal are not necessarily independent asinteractions can and probably do occur [52 53]

The sample and the reagent could be either in continuousflow or in stopped flow approach Continues flow CL systemsare used in gas phase and solution phase reactions Thesample and CL reagents are continuously pumped andmixedtogether using amerging zones of flow injectionmanifold andsent to a flow cell or mixed and the emission in an integralreactor flow cellThe signal is observed when the cell is totallyfilled with the reactionmixture at a fixed period aftermixingIn this case it is possible to obtain a constant and reproduciblesignal easier to measure which represents the integratedoutput over the residence time of the reaction mixture inthe cell Optimum sensitivity is achieved by adjusting theflow rate the observation cell volume and transfer channelvolumes with the aim of observing portion of emissionprofile to occur at the maximum of the CL intensity- versustime profile Disadvantage of this approach include the highreagent consumption and the fact that no kinetic informationis obtained and that only a very small portion of the total CLintensity is measured for slow reactions (Figure 5)

Stopped flow approach has interesting features for CLmonitoring in very fast CL reactions In this case the sampleand reagent are efficiently mixed in a reaction chamber and

ISRN Spectroscopy 9

Detector

Reaction cell

(a) (b) (c)

Or area in At

Relative units

ConcentrationReaction time (s)

t0 t1

ICL

Imax

Max(t1)

Figure 5 Basic steps in a CL process (a) the sample and reagent(s) are introduced is the reaction cell and the final reagent is injected toinitiate the CL emission then lights is monitored by the detector (b) curve showing CL intensity as a function of time after reagents mixingto initiate the reaction (the decay of the signal is due to the consumption of reagents and changes in the CL quantum efficiency with time)(c) a calibration function is established in relation to increasing analyte concentrations

rapidly forced to an observation cell in which the flow isviolently stopped allowing monitoring of the full intensity-time curve and allowing kinetics measurements (Figure 5)which can be related to the analyte concentration with ahigher precision and selectively than using peak height orareas

311 Reaction Cell Depending on the CL reaction that iscarried out in liquid solid or gas phases it is possible to usedifferent reaction cells the essential requirements for theirdesign being that the maximum intensity must be reachedwhile the stream containing the analyte and the CL reagentsis in front of the detector It is a very critical variable becausein both static and flow methods the magnitude of the CLintensity is proportional to the volume of the cell Usually itis possible to apply any material that transmits light in thevisible range and that is compatible with the sample such asglass quartz or even acrylic or other plastics Polystyrene isnot recommended because it can build up static electricitywhich may contribute to elevated background levels Moretransparent cell materials allow more light to reach thedetector resulting in better detection limits

In the case of flow CL measurements in the liquid phasethe cell design most frequently used is a long spiral cell(about 1 cm) which is located as close to the detector aspossible The large volume is required due to the need tocollect a greater amount of emitted light and because the thinsize required (50ndash100120583m) minimizes band broadening andprovides a thin optical path to reduce inner filtering effectsof the CL emission With the purpose of minimize sampledispersion usually the injection valve and the cell must beplaced as close to reach the other ones as possible

In heterogeneous systems the reagents can be immobi-lized on a solid surface either covalently behind amembrane

or by absorption and it can interact with the analyte bydiffusion or convection In gas phase CL reaction the sampleand the CL reagents flow inside a reactor through calibratedleaks and the CL emission produced and monitored bythe detector through and optical window Commercial andhomemade luminormeters for liquid solid and gas phaseanalysis use various cell configurations

312 Detector There are four basic requirements for thedetectors Which constitute the critical parts of the lumi-normeter

(1) It must be able to detect a light signal over severalorders of magnitude of intensity form just a fewphotons per second to tens of millions photons persecond

(2) It must be sensitive at least over the spectral range400ndash600 nm and ideally over the complete range ofthe visible spectrum (ie 380ndash750 nm) and even overthe UV and IR regions When its sensitivity varieswith wavelength as is usually the case it must bepossible to correct the data in this respect

(3) The signal output presented by the detector shouldbe directly related to the light intensity reaching itideally linearly over the entire sensitively range Thesignal from the detector should be produced in a formthat can be easily displayed recorded and analyzed

(4) The speed of response of the detector must be muchfaster than the rate of the CL reaction if the signaloutput is not to be a distorted version of the truesignal

Light is detected by photosensitive devices through thegeneration of photocurrent Photomultiplier tubes (PMT) are

10 ISRN Spectroscopy

the detection devices more frequently used in CL becauseof their high gains making them suitable for low light leveldetection However they must be operated with very stablepower supplies to keep the total gain constant There are twodifferent configurations for PMTs depending on their posi-tions in relation to the reaction cell either to the side (ldquoside-onrdquo configuration) or underneath the reaction cell (ldquoend-onrdquoconfiguration) Side on PMTs is usually more economicalthan end on models and their vertical configuration takes upless space than be end on version However end-on PMTshave large photocathode area and offer greater uniformity oflight collection and of the response

Also in the absence of light stimulation all practicallight detectors produce an unwanted output signal termedldquobackgroundrdquo which is referred to as ldquodark countsrdquo or darkcurrentrdquo depending on the applications At moderate biasvoltages dark current can be due primarily to thermalemission of electrons at the photocathode and from thedynodes This undesirable signal can be reduced by coolingthe PMT using temperature of 60 to 0∘C

32 Pharmaceutical Application of Chemiluminescence Reac-tion From Table 1 one can observe that varsitile compoundsand pharmaceuticals could be directly or indirectly quanti-fied For the indirect method of quantification either chemi-cally derived so as to exhibit the compound luminescent effector by measuring the compounds quenching effect

33 Chemiluminescent Immunoassay One of the most sig-nificant applications of chemiluminescent molecules hasbeen labels in immunoassay Not unexpectedly the con-straints arise from the need to couple the chemiluminescentmolecules to the analytes of respective antibodies in sucha way that the properties of the label are not disturbed Inparticular aminobutyl ethyl isoluminol (ABEl) which can becoupled to low molecular weight compounds without loss inquantum yield

As a result ABEl and relatedmolecules have been used aslabels for a variety of steroid immunoassays which have levelsof performance comparable to those of the correspondingradioimmunoassay A universal problem of these methodsarises from the extensive quenching of the signal whenthe label is associated with antibody So great is the lossin quantum yield under these conditions that dissociationof the complex prior to luminometry has been adoptedas an obligatory step As indicated above the majority ofchemiluminescent hapten immunoassays so far describedhave been based on ABEl or similar labels These assaysinclude plasma estradiol-l7 testosterone and progesteroneas well as urinary conjugated steroids such as pregnanediol-3n-glucuronide and estrone glucuronide Dissociation of thelabel prior to signal detection has been facilitated by meansof strong alkali often at high temperatureThough this resultsin satisfactory light emission it does introduce a step intothe analytical procedure which is operationally complex andwhich may affect the overall performance of the assay itselfIt is possible that acridinium esters will provide simpleralternatives though as yet there is only one report of their use

on steroid immunoassay In this case a synthetic acridiniumderivative of estradiol was prepared which could be detectedwith high sensitivity Assay performancewas however limitedby the low affinity of the antibody for the labelled derivativeonce again illustrating the dependence of hapten systems onsuitable immunochemistry

The procedure has centred on the development of assaymethods for polypeptides using acridinium ester labelledantibodies It has employed almost exclusively two-site assayprocedures in which the analyte is reacted either sequentiallyor simultaneously with labeled antibodies and solid-phaseantibodies In this procedure the uptake of labelled antibodyon to solid-phase antibody is a function of the quantity of ana-lyte bound and hence the analyte concentration in the sampleThe most satisfactory solid-phase antibodies developed sofar are prepared by reacting antibody preparations with thediazonium salt of re-precipitated aminoaryl cellulose Thispreparation has the advantage that it remains in suspensionduring the reaction without agitation It is also extremelyefficient as an immune adsorbent since it has a high bindingcapacity for protein as well as very low non-specific uptake ofprotein [54 55]

In this procedure standards or serum samples (100 pi) arereacted with a monoclonal antibody (100 p1 15 ng) labelledwith 3molmol acridinium ester After 2 h solid-phase anti-body suspension (100 p1 50 Pg) is added and after a further1 h 1mL of buffer is added and the mixture centrifugedAfter one further 1mL wash the chemiluminescent activityassociated with the solid-phase is quantified luminometri-cally following injection of 200pl water to resuspend thepellet followed by 200 p1 01 (vv) H

2

O2

in 01M NaOHThe photon counts are integrated over a 2 s period and thebound counts related to the dose of analyte present The useof reagent excess methodology together with an acridiniumester label of high specific activity combine to make this themost sensitive immunoassay yet described for TSH [56 57]

From the clinical point of view this assay offers a remark-able breakthrough in diagnostic and therapeutic testingPituitary TSH secretion plays a central role in the control ofthyroid function Since it is under negative feedback controlby the thyroid hormones whose production it stimulates itprovides a sensitive indicator of thyroid status

The main applications of TSH measurement being in theconfirmation of hypothyroidismwhere levels are elevated andin monitoring the pituitary response to thyrotropin releasehormone (TRH) This immune-chemiluminometric assay(ICMA) is at present under evaluation [58]

References

[1] L Boslashtter-Jensen ldquoLuminescence techniques instrumentationand methodsrdquo Radiation Measurements vol 27 no 5-6 pp749ndash768 1997

[2] Y Hajime Nitride Phosphors and Solid-State Lighting Taylor ampFrancis 2011

[3] K H Drexhage ldquoInfluence of a dielectric interface on fluores-cence decay timerdquo Journal of Luminescence vol 1-2 no C pp693ndash701 1970

ISRN Spectroscopy 11

[4] B Wen Thermally Stimulated Luminescence Excitation Spec-troscopy as a Technique to Measure the Photoionization Energyof Sr(SCN)

2

Eu2+ vol 2004 Maureen Grasso Dean of theGraduate School The University of Georgia

[5] F McElroy EPANERL Determination of Ozone by UltravioletAnalysis May 2000

[6] C-Y Liu andA J Bard ldquoElectrochemistry and electrogeneratedchemiluminescence with a single faradaic electroderdquoAnalyticalChemistry vol 77 no 16 pp 5339ndash5343 2005

[7] A Pavlova P Ivanova and T Dimova ldquoSulfur compounds inpetroleum hydrocarbon streamsrdquo PetroleumampCoal vol 54 no1 pp 9ndash13 2012

[8] A A Alwarthan and F A Aly ldquoChemiluminescent determi-nation of pyridoxine hydrochloride in pharmaceutical samplesusing flow injectionrdquo Talanta vol 45 no 6 pp 1131ndash1138 1998

[9] C Fu-Chun H Yu-Ming and Z Zhun-Jun ldquoChemilumi-nescent determination on a luminol reaction of streptomycinsulfate by flow injection analysisrdquo Journal of Southwest ChinaNormal University vol 2004 no 1 pp 79ndash83 2004

[10] J K Robinson M J Bollinger and J W Birks ldquoLuminolH2

O2

chemiluminescence detector for the analysis of nitric oxide inexhaled breathrdquo Analytical Chemistry vol 71 no 22 pp 5131ndash5136 1999

[11] H Sun J Wang P Chen and T Wang ldquoDetermination ofofioxacin and levofloxacin in pharmaceutical preparation andhuman urine using a new flow-injectionrdquo Asian Journal ofPharmaceutical Sciences and Research vol 1 no 5 pp 21ndash322011

[12] P Thongsrisomboon B Liawruangrath S Liawruangrath andS Satienperakul ldquoDetermination of nitrofurans residues inanimal feeds by flow injection chemiluminescence procedurerdquoFood Chemistry vol 123 no 3 pp 834ndash839 2010

[13] C Yu Y Tang X Han and X Zheng ldquoSensitive assay forcatecholamines in pharmaceutical samples and blood plasmausing flow injection chemiluminescence analysisrdquo AnalyticalSciences vol 22 no 1 pp 25ndash28 2006

[14] W Freyer C C Neacsu and M B Raschke ldquoAbsorptionluminescence and Raman spectroscopic properties of thinfilms of benzo-annelated metal-free porphyrazinesrdquo Journal ofLuminescence vol 128 no 4 pp 661ndash672 2008

[15] C J Borman B P Sullivan CM Eggleston and P J S ColbergldquoThe use of flow-injection analysis with chemiluminescencedetection of aqueous ferrous iron in waters containing highconcentrations of organic compoundsrdquo Sensors vol 9 no 6 pp4390ndash4406 2009

[16] F A Aly N A Alarfaffj and A A Alwarthan ldquoPermanganate-based chemiluminescence analysis of cefadroxil monohydratein pharmaceutical samples and biological fluids using flowinjectionrdquo Talanta vol 47 no 2 pp 471ndash478 1998

[17] F A Aly S A Al-Tamimi and A A Alwarthan ldquoChemilu-minescence determination of some fluoroquinolone derivativesin pharmaceutical formulations and biological fluids using[Ru(bipy) 2+

3

]-Ce(IV) systemrdquo Talanta vol 53 no 4 pp 885ndash893 2001

[18] Y-H Li ldquoChemiluminescence determination of sulpiride inpharmaceutical preparations and biological fluids based onKMnO

4

-tween 80 reactionrdquo Journal of the Chinese ChemicalSociety vol 56 no 1 pp 164ndash168 2009

[19] A Penzkofer and Y Lu ldquoFluorescence quenching of rhodamine6G in methanol at high concentrationrdquo Chemical Physics vol103 no 2-3 pp 399ndash405 1986

[20] C Dodeigne L Thunus and R Lejeune ldquoChemiluminescenceas a diagnostic tool A reviewrdquo Talanta vol 51 no 3 pp 415ndash439 2000

[21] J Hodak D Harvey and B Valeur Introduction to Fluorescenceand Phosphorescence 2008

[22] E L Wehry ldquoMolecular fluorescence phosphorescence andchemiluminescence spectrometryrdquo Analytical Chemistry vol52 no 5 1980

[23] M Sauer J Hofkens and J EnderleinHandbook of FluorescenceSpectroscopy and Imaging WILEY-VCH Verlag GmbH amp CoKGaA Weinheim Germany 2011

[24] J Han J Jose E Mei and K Burgess ldquoChemiluminescentenergy-transfer cassettes based on fluorescein and nile redrdquoAngewandte Chemie International Edition vol 46 no 10 pp1684ndash1687 2007

[25] A M Garcıa-Campana and W R G Baeyens ldquoPrinciples andrecent analytical applications of chemiluminescencerdquo Analusisvol 28 no 8 pp 686ndash698 2000

[26] A M Jimenez and M J Navas ldquoChemiluminescence methods(present and future)rdquo Grasas y Aceites vol 53 no 1 pp 64ndash752002

[27] Miyawa and Schulman ldquoHandbook of Pharmaceutical analy-sisrdquo in Luminescence Spectroscopy pp 457ndash460

[28] R J Stanley B King and S G Boxer ldquoExcited state energytransfer pathways in photosynthetic reaction centers 1 Struc-tural symmetry effectsrdquo Journal of Physical Chemistry vol 100no 29 pp 12052ndash12059 1996

[29] F Mccapra The Chemiluminescence of Organic CompoundsSchool of Molecular Sciences University of Sussex FalmerBrighton UK

[30] G S Denisov N S Golubev V M Schreiber S S Shajakhme-dov and A V Shurukhina ldquoEffect of intermolecular hydrogenbonding and proton transfer on fluorescence of salicylic acidrdquoJournal of Molecular Structure vol 436-437 pp 153ndash160 1997

[31] H S Lee Y S Lee and G-H Jeung ldquoPotential energy surfacesfor lith and photochemical reactions Lilowast + H2 harr LiH + HrdquoJournal of Physical Chemistry A vol 103 no 50 pp 11080ndash110881999

[32] M Anpo and M Che ldquoApplications of photoluminescencetechniques to the characterization of solid surfaces in relation toadsorption catalysis and photocatalysisrdquoAdvances in Catalysisvol 44 pp 119ndash257 1999

[33] C Jan Hummelen T M Luider D Oudman J N Koek andH Wynberg 12-Dioxetanes Luminescent and NonluminescentDecomposition Chemistry and Potential Applications StateUniversity of Groningen Groningen The Netherlands

[34] E F Fabrizio A Payne N E Westlund A J Bard andP P Magnus ldquoPhotophysical electrochemical and electro-generated chemiluminescent properties of 910-dimethyl-712-diphenylbenzo[k]fluoranthene and 910-dimethylsulfone-712-diphenylbenzo[k]fluoranthenerdquo Journal of Physical ChemistryA vol 106 no 10 pp 1961ndash1968 2002

[35] B Valeur D Harvey and J Hodak Molecular FluorescencePrinciples and Applications Wiley 2001

[36] V Narasimham and N Modeling Analysis of Chemilumi-nescence Sensing for Syngas Methane and Jet-a CombustionGeorgia Institute of Technology 2008

[37] K Thompson and J Rodriguez The Behavior of Photo-ExcitedMolecules Biological Actions and Biotechnological Applications2008


[38] I Baraldi G Brancolini F Momicchioli G Ponterini andD Vanossi ldquoSolvent influence on absorption and fluorescencespectra of merocyanine dyes a theoretical and experimentalstudyrdquo Chemical Physics vol 288 no 2-3 pp 309ndash325 2003

[39] R-I Tigoianu A Airinep and D-O Dorohoi ldquoSolvent influ-ence on the electronic fluorescence spectra of anthracenerdquoRevista de Chimie vol 61 no 5 pp 491ndash494 2010

[40] P Hrdlovic J Donovalova H Stankovicova and A GaplovskyldquoInfluence of polarity of solvents on the spectral properties ofbichromophoric coumarinsrdquoMolecules vol 15 no 12 pp 8915ndash8932 2010

[41] P Anger P Bharadwaj and L Novotny Enhancement andQuenching of Single-Molecule FluorescenceTheAmerican Phys-ical Society The Institute of Optics and Department of Physicsand Astronomy University of Rochester Rochester NY USAMarch 2006

[42] A Mark Behlke L Huang L Bogh S Rose and E J DevorFluorescence Quenching by Proximal G-bases Integrated DNATechnologies 2005

[43] M Mimuro ldquoVisualization of excitation energy transfer pro-cesses in plants and algaerdquo Photosynthesis Research vol 73 no1ndash3 pp 133ndash138 2002

[44] D Nolting T Schultz I V Hertel and R Weinkauf ldquoExcitedstate dynamics and fragmentation channels of the protonateddipeptide H2N-Leu-Trp-COOHrdquo Physical Chemistry ChemicalPhysics vol 8 no 44 pp 5247ndash5254 2006

[45] D Kimbrough Chemiluminescence and Anti-Oxidants 2000[46] H Sun Cho D H Jeong M-C Yoon et al ldquoExcited-state

energy transfer processes in phenylene- and biphenylene-linkedand directly-linked zinc(II) and free-base hybrid diporphyrinsrdquoJournal of Physical Chemistry A vol 105 no 17 pp 4200ndash42102001

[47] K Zargoosh M J Chaichi S Asghari M Qandalee and MShamsipur ldquoA study of chemiluminescence from reaction ofbis(246-trichlorophenyl) oxalate hydrogen peroxide anddiethyl-2-(cyclohexylamino)-5-[(E)-2-phenyl-1-ethenyl]-34-furandicarboxylate as a novel fluorescerrdquo Journal of the IranianChemical Society vol 7 no 2 pp 376ndash383 2010

[48] MG Prisant C T Rettner and R N Zare ldquoA direct interactionmodel for chemiluminescent reactionsrdquoThe Journal of ChemicalPhysics vol 81 no 6 pp 2699ndash2712 1984

[49] M J Navas and AM Jimenez ldquoChemiluminescent methods inolive oil analysisrdquo Journal of the American Oil Chemistsrsquo Societyvol 84 no 5 pp 405ndash411 2007

[50] K-T Wong T-H Hung T-C Chao and T-I Ho ldquoSynthesisproperties and electrogenerated chemiluminescence (ECL) ofa novel carbazole-based chromophorerdquoTetrahedron Letters vol46 no 5 pp 855ndash858 2005

[51] J Yu L Ge P Dai C Zhan S Ge and J Huang ldquoA novelglucose chemiluminescence biosensor based on a rhodaninederivative chemiluminescence system and multilayer-enzymemembranerdquo Turkish Journal of Chemistry vol 34 no 4 pp489ndash498 2010

[52] ACL Instruments Inc Chemiluminescence Instrument Hard-ware Basic Configuration

[53] T Peter So and C Y Dong Fluorescence Spectrophotome-try Encyclopedia of Life Sciences Macmillan Publishers LtdNature Publishing 2002

[54] W R G Baeyens S G Schulman A C Calokerinos et alldquoChemiluminescence-based detection principles and analyticalapplications in flowing streams and in immunoassaysrdquo Journal

of Pharmaceutical and Biomedical Analysis vol 17 no 6-7 pp941ndash953 1998

[55] C Wang J Wu C Zong J Xu and H-X Ju ldquoChemilumi-nescent immunoassay and its applicationsrdquo Chinese Journal ofAnalytical Chemistry vol 40 no 1 pp 3ndash10 2012

[56] M Hao and Z Ma ldquoAn ultrasensitive chemiluminescencebiosensor for carcinoembryonic antigen based on autocatalyticenlargement of immunogold nanoprobesrdquo Sensors vol 12 pp17320ndash17329 2012

[57] W P Collins Complementary Immunoassays John Wiley ampSons 1988

[58] E Bolton andM M Richter ldquoChemiluminescence of Tris(221015840-bipyridyl)ruthenium(II) a glowing experiencerdquo Journal ofChemical Education vol 78 no 1 pp 47ndash48 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy



Analytical ChemistryVolume 2014

Journal of


Quantum Chemistry


Organic Chemistry International


CatalystsJournal of

ElectrochemistryInternational Journal of


2 ISRN Spectroscopy

Singlet excited states

Internalconversion

Vibrationalrelaxation

Ener

gy

Groundstate

Absorption Fluorescence

Triplet excited state

Intersystemcrossing

Internaland

externalconversion

Phosphorescence

T1

1205822 12058211205823120582r 1205824

120582998400

r

S1

S2

S0Vibrationalrelaxation

Figure 1 Jablonski diagram describing the electronic levels of common organic molecules and possible transitions between different singletand triplet states (M Sauer J Hofkens and J Enderlein Handbook of Fluorescence Spectroscopy and Imaging)

to as intersystem crossing and is a spin-dependent internalconversion process A molecule in excited triplet state maynot always use intersystem crossing to return to the groundstate It could loss energy by emission of a photon the processcalled phosphorescence A triplet-singlet transition is muchless probable than a singlet-singlet transition The lifetime ofthe excited triplet state can be up to 10 sec in comparisonwith 10minus5 s to 10minus9 s of average lifetime of an excited singletstate Emission from triplet-singlet transition can continueafter initial irradiation [19 20]

2 The Nature of ChemiluminescenceReactions

In general chemiluminescence reaction yields one of thereaction products in an electronic excited state producinglight on falling to the ground state As can be seen in Figure 1the process of light emission in chemiluminescence is thesame as in photoluminescence except for the excitation pro-cess In fluorescence and phosphorescence the electronicallyexcited state is produced by absorption of Uv-visible lightreturning to the ground state (S

0

) from the lowest singletexcited state (S

1

) or from the triplet excited state (T1

) [21 22]In general a chemiluminescent reaction can be generated

by two basic mechanisms (Figure 2) in a direct reactiontwo reagents usually a substrate and an oxidant in thepresence of some cofactors react to from a product orintermediate sometimes in the presence of a catalyst Thensome fraction of the product or intermediate will be formedin an electronically excited state which can subsequentlyrelax to the ground state with emission of a photon Thesubstrate is the CL precursors which is converted into the

electronically excitedmolecule responsible for light emissionor acting as the energy as transfer donor in indirect CL Thecatalyst enzyme or metal ions reduce the activation energyand provide an adequate environment for producing highCL efficiency out of the process Cofactors sometimes arenecessary to convert one ormore of the substrates into a formcapable of reacting and interacting with the catalyst or toprovide an efficient leaving group if bond cleavage is requiredto produce the excited emitter On the contrary indirect orsensitized CL is based on a process of transfer of energy ofthe excited speciersquos to a fluorophore [23] This process makesit possible for those molecules that are unable to be directlyinvolved in CL reaction to transfer their excess of energy toa fluorophore that in turn is excited releasing to its groundstate with photon emission All of these paths lead to a greatvariety of practical uses of CL in solid gas and liquid phases[24]

21 Requirements for Chemiluminescence Emission For achemical reaction to produce light it should meet someessential requirements

(1) The reactionmust be exothermic to produce sufficientenergy to form the electronically excited state Topredict whether the CL reaction will occur or not itis possible to use free energy (Δ119866)

Δ119866 = Δ119867 minus 119879Δ119878 (1)

where 119879 is temperature Δ119867 is enthalpy and Δ119878 isentropy

ISRN Spectroscopy 3


(Cofactors)


Catalyst


precursor)

F + h

P + h

P + h




119860



minus Δ119867119864

ge Δ119864EX (2)


Δ119866 geℎ119888

120582ex=286 times 10

4

120582ex (3)



AB + D

D

BA

(6)

(5)

(7)

ABlowast

lowastA

A + B

BF +

ACC

+ B

AB + F

(3)

(2)

(4)

AB + light







4 ISRN Spectroscopy









4


4119894


4

lt Δ1198674119894


Pote

ntia

l ene

rgy l

evel


ΔH

ΔH








ISRN Spectroscopy 5



119889

) [35]









6 ISRN Spectroscopy







6


[8ndash13]



[14 15]



[10 12]


4





119902


OslashOslash119902

=1

1 + 119896119876

(pound119902

) [119876]

(5)

where 119896119876


119902



119902





119870ET =1

pound119863

1198770

119877 (6)


119863


ISRN Spectroscopy 7


0





= 119896119891

+ 119896119889




times Oslash119864

times Oslash119891

(7)

where Oslash119877


119864


119891


In type II reaction


times Oslash119864

times Oslash119891

times OslashET (8)








8 ISRN Spectroscopy













ISRN Spectroscopy 9

Detector

Reaction cell

(a) (b) (c)

Or area in At

Relative units


t0 t1

ICL

Imax

Max(t1)






















2

O2




References






2








O2









3



4





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of



ISRN Spectroscopy 3


(Cofactors)


Catalyst


precursor)

F + h

P + h

P + h




119860



minus Δ119867119864

ge Δ119864EX (2)


Δ119866 geℎ119888

120582ex=286 times 10

4

120582ex (3)



AB + D

D

BA

(6)

(5)

(7)

ABlowast

lowastA

A + B

BF +

ACC

+ B

AB + F

(3)

(2)

(4)

AB + light







4 ISRN Spectroscopy









4


4119894


4

lt Δ1198674119894


Pote

ntia

l ene

rgy l

evel


ΔH

ΔH








ISRN Spectroscopy 5



119889

) [35]









6 ISRN Spectroscopy







6


[8ndash13]



[14 15]



[10 12]


4





119902


OslashOslash119902

=1

1 + 119896119876

(pound119902

) [119876]

(5)

where 119896119876


119902



119902





119870ET =1

pound119863

1198770

119877 (6)


119863


ISRN Spectroscopy 7


0





= 119896119891

+ 119896119889




times Oslash119864

times Oslash119891

(7)

where Oslash119877


119864


119891


In type II reaction


times Oslash119864

times Oslash119891

times OslashET (8)








8 ISRN Spectroscopy













ISRN Spectroscopy 9

Detector

Reaction cell

(a) (b) (c)

Or area in At

Relative units


t0 t1

ICL

Imax

Max(t1)






















2

O2




References






2








O2









3



4





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of



4 ISRN Spectroscopy









4


4119894


4

lt Δ1198674119894


Pote

ntia

l ene

rgy l

evel


ΔH

ΔH








ISRN Spectroscopy 5



119889

) [35]









6 ISRN Spectroscopy







6


[8ndash13]



[14 15]



[10 12]


4





119902


OslashOslash119902

=1

1 + 119896119876

(pound119902

) [119876]

(5)

where 119896119876


119902



119902





119870ET =1

pound119863

1198770

119877 (6)


119863


ISRN Spectroscopy 7


0





= 119896119891

+ 119896119889




times Oslash119864

times Oslash119891

(7)

where Oslash119877


119864


119891


In type II reaction


times Oslash119864

times Oslash119891

times OslashET (8)








8 ISRN Spectroscopy













ISRN Spectroscopy 9

Detector

Reaction cell

(a) (b) (c)

Or area in At

Relative units


t0 t1

ICL

Imax

Max(t1)






















2

O2




References






2








O2









3



4





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of



ISRN Spectroscopy 5



119889

) [35]









6 ISRN Spectroscopy







6


[8ndash13]



[14 15]



[10 12]


4





119902


OslashOslash119902

=1

1 + 119896119876

(pound119902

) [119876]

(5)

where 119896119876


119902



119902





119870ET =1

pound119863

1198770

119877 (6)


119863


ISRN Spectroscopy 7


0





= 119896119891

+ 119896119889




times Oslash119864

times Oslash119891

(7)

where Oslash119877


119864


119891


In type II reaction


times Oslash119864

times Oslash119891

times OslashET (8)








8 ISRN Spectroscopy













ISRN Spectroscopy 9

Detector

Reaction cell

(a) (b) (c)

Or area in At

Relative units


t0 t1

ICL

Imax

Max(t1)






















2

O2




References






2








O2









3



4





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of



6 ISRN Spectroscopy







6


[8ndash13]



[14 15]



[10 12]


4





119902


OslashOslash119902

=1

1 + 119896119876

(pound119902

) [119876]

(5)

where 119896119876


119902



119902





119870ET =1

pound119863

1198770

119877 (6)


119863


ISRN Spectroscopy 7


0





= 119896119891

+ 119896119889




times Oslash119864

times Oslash119891

(7)

where Oslash119877


119864


119891


In type II reaction


times Oslash119864

times Oslash119891

times OslashET (8)








8 ISRN Spectroscopy













ISRN Spectroscopy 9

Detector

Reaction cell

(a) (b) (c)

Or area in At

Relative units


t0 t1

ICL

Imax

Max(t1)






















2

O2




References






2








O2









3



4





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of



ISRN Spectroscopy 7


0





= 119896119891

+ 119896119889




times Oslash119864

times Oslash119891

(7)

where Oslash119877


119864


119891


In type II reaction


times Oslash119864

times Oslash119891

times OslashET (8)








8 ISRN Spectroscopy













ISRN Spectroscopy 9

Detector

Reaction cell

(a) (b) (c)

Or area in At

Relative units


t0 t1

ICL

Imax

Max(t1)






















2

O2




References






2








O2









3



4





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of



8 ISRN Spectroscopy













ISRN Spectroscopy 9

Detector

Reaction cell

(a) (b) (c)

Or area in At

Relative units


t0 t1

ICL

Imax

Max(t1)






















2

O2




References






2








O2









3



4





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of



ISRN Spectroscopy 9

Detector

Reaction cell

(a) (b) (c)

Or area in At

Relative units


t0 t1

ICL

Imax

Max(t1)






















2

O2




References






2








O2









3



4





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of












2

O2




References






2








O2









3



4





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of





2








O2









3



4





















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of



































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of












Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy




Journal of


Quantum Chemistry




CatalystsJournal of



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A Recent Review on Chemiluminescence Reaction, Principle ...downloads.hindawi.com/journals/isrn.spectroscopy/2013/230858.pdf · A Recent Review on Chemiluminescence Reaction, Principle