LUMINESCENCE / Overview 305
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Lockley WJS (1989) Regiochemical differences in the
isotopic fractionation of deuterated benzoic acid
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LOW ENERGY ELECTRON DIFFRACTION
See SURFACE ANALYSIS: Low Energy Electron Diffraction
LUMINESCENCE
Contents
Overview
Solid Phase
Overview
N W Barnett and P S Francis, Deakin University,
Geelong, VIC, Australia
& 2005, Elsevier Ltd. All Rights Reserved.
Introduction
The observation and investigation of luminescent
phenomena has a long and delightful history and
some of the more important milestones are briefly
described here. Naturally occurring luminescence,
such as fireflies, St Elmo’s fire, and shining flesh has
fascinated humans since the dawn of time, with an
early record appearing in Chinese literature between
1500 and 1000 BC. A millennium or so later, Aristotle
(384–322 BC) astutely noted that these emanations
were produced without heat. Subsequently, Caius
Plinius Secundus (Pliny the Elder, AD 23–79) de-
scribed, in detail, a number of luminous organisms.
Notwithstanding the revolutionary nature of these
observations of bioluminescence, made by two such
great philosophers, a rigorous scientific approach to
the subject was not taken until the mid-sixteenth
century. This study culminated in the publication, in
1555, of a book by Conrad Gesner (1516–1565)
concerned solely with luminescence. However, Sir
Tanaka N and Thornton (1977) Structural and isotopic
effects in hydrophobic binding measured by HPLC. A
stable and highly precise model for hydrophobic inter-
action in biomembranes. Journal of the American Chem-
ical Society 99: 7300–7306.
Wade D (1999) Deuterium isotope effects on noncovalent
interaction between molecules. Chemico-Biological
Interactions 117: 191–217.
Robert Boyle (1627–1691), son of the Earl of Cork,
who came to be known as the father of analytical
chemistry, first categorized the essential differences
between incandescence and luminescence in 1668.
The following year saw the first artificial lumines-
cence, which accompanied Hennig Brandt’s dis-
covery and isolation of elemental phosphorus.
More than three centuries passed before the emana-
tions from white phosphorus were correctly charac-
terized as chemiluminescence. Cambridge professor
and president of the Royal Society, Sir George Gab-
riel Stokes (1819–1903), was the first to characterize
the bichromatic nature of crystal fluospar as a true
emission (actually phosphorescence) in 1845. He
also coined the term fluorescence as an analogy to
opalescence and noted that in the production of flu-
orescence the absorption of shorter wavelengths re-
sulted in emission at longer wavelengths (Stokes’
law). In 1877, Raziszewski was probably the first to
observe chemiluminescence from synthetically pro-
duced organic compounds during the preparation of
lophine (2,4,5-triphenylimidazole) from hydrobenz-
amide.
The earliest usage of the word luminescence is
credited to Eilhardt Wiedemann (1852–1928), who
in 1888 used the term to describe the emission of
light that was not the result of a rise in temperature
(‘cold light’). He defined six classes of luminescence
by the source of energy that stimulated the emission.
306 LUMINESCENCE / Overview
The present number of classes of luminescent phe-
nomena is somewhere around 20; however, the
boundaries between some of these classifications is
more than a little blurred. In stark contrast to in-
candescence, luminescent phenomena are not only
cold but are also of relatively low intensity, for
example:
* the mysterious St Elmo’s Fire and aurora australis
(electro- or radioluminescence);
* emission from rubbing or shattering crystals
(triboluminescence);
* the ephemeral blue haze from a glass of gin and
tonic in the sunlight (fluorescence);
* the enchanting emanations from ‘lightening bugs’
(fireflies) on a summer evening (chemilumines-
cence).
Luminescence is generally less intense than incan-
descence, but it often emanates from extremely small
amounts of matter, which has beneficial implications
for analytical science. Nevertheless, the utilization of
luminescence for analysis is quite a recent innovat-
ion. The following commentary describes the fun-
damental spectroscopic and chemical principles un-
derlying luminescence in relation to its application in
analytical science. As other articles will deal with
atomic spectroscopy, this discussion will be restricted
to analytical molecular luminescence spectroscopy
including fluorescence, phosphorescence, and chemi-
luminescence (bioluminescence being a special case
of chemiluminescence).
By monitoring the relative intensity of lumines-
cence as a function of concentration it is possible to
quantitatively determine (often at trace levels) a
range of inorganic and organic analytes. Contem-
porary analytical texts reveal that fluorimetric
methodology is far more commonplace than either
phosphorescence or chemiluminescence. The re-
search literature, however, indicates an increased ac-
ceptance of the latter two spectroscopies particularly
when combined with either organized media or flow
analysis, respectively. Luminescence methodology
generally offers superior selectivity, detectability,
and linear calibration range compared to that at-
tainable with absorption spectrometry. Unfortunate-
ly, there are a relatively limited number of molecules
that will exhibit luminescence thus restricting its ap-
plicability compared to absorption techniques. Al-
though all forms of luminescence have a common
quantum mechanical basis for emission, the route to
the excited state defines their respective class. Given
that the theory of photoluminescence is better un-
derstood than that of the various related phenomena,
a basic synopsis of fluorescence and phosphorescence
is presented prior to an introduction to chemically
induced luminescence.
Photoluminescence
General Principles
Photoluminescence occurs as the result of electronic
excitation within a molecule brought about by
the absorption of a photon. The question therefore
arises, as the vast majority of molecules do not lu-
minescence, what are the quantum mechanisms that
give rise to fluorescence and phosphorescence and
how does molecular structure aid or inhibit these
processes? Figure 1 is a Jabłoński diagram, named in
honor of the Ukrainian born physicist, Professor
Alexander Jabłoński (1898–1980), who is considered
by many to be the father of fluorescence spectros-
copy. These schematic energy level diagrams are
useful tools for the understanding of molecular elec-
tronic excitation and deexcitation leading to photon
emission. The first observation to be made from
Figure 1 is that fluorescence emission (F) results from
an electron falling from the lowest vibrational level
of an electronically excited state (S1) to any of the
vibrational excited levels of the ground state (S0).
Phosphorescence emission (P) is a similar process
except that the upper electronic state is a triplet (T1).
The terms singlet and triplet refer to the relative spins
of the electrons in the ground and excited states.
When the spins are paired (antiparallel) the upper
level is termed a singlet, and when the spins are
unpaired (parallel) a triplet state exists. The nomen-
clature arises from the observed multiplicity in spec-
tra measured under the influence of a magnetic field.
It is noteworthy that T1 states are less energetic than
S1; this is a direct consequence of electrons with par-
allel spins being further apart thus exhibiting less
mutual repulsion (spin correlation, Hund’s rule).
Hence, for a given compound the phosphorescence
emission will occur at longer wavelengths than the
fluorescence. The photo-induced promotion of an
electron from S0 directly to T1 has not been shown in
Figure 1 as the simultaneous change of molecular
orbital and electronic spin has a very low probability
of occurrence. In fact, such transitions are often re-
ferred to as ‘forbidden’ by the spin selection rule.
As most compounds are not luminescent, this im-
plies that there must be more efficient alternative
deexcitation mechanisms available to molecules in S1
or T1 states to return to S0 other than ejection of a
photon. To understand the nature of the spec-
troscopic processes shown in Figure 1, their relative
rates of occurrence and the influences on the various
pathways must be considered.
 LUMINESCENCE / Overview 307

Singlet states

Triplet states
S2 8 3
IC 7 2
6 T2 1
5
VR 4
3
2
1 7
S1 v ′=0 ISC 6
5
4
3
2
1
T1 v ′=0
IC
A

A F

P
EC/IC

5
4
VR 3
2
1
S0 v ″=0

Figure 1 A Jabłoński diagram for a hypothetical luminescent molecule, where A is absorption, F is fluorescence, P is phospho-
rescence, VR is vibrational relaxation, IC is internal conversion, EC is external conversion, ISC is intersystem crossing, v00 and v0 are
vibrational levels associated with each electronic state, S0 is the electronic ground state, S1 and S2 are excited singlet states and T1
and T2 are excited triplet states.

At room temperature in solution, we may assume
that all molecules will be in the lowest vibrational
level (v00 ¼ 0) of the ground state (S0), thus absorption
will originate solely from v00 ¼ 0. According to the
Franck–Condon principle, the initial step (absorption
of a photon) is extremely rapid, requiring only
10
15–10
14 s. Immediately after absorption; either
of the excited singlets (S1 or S2) will undergo vibra-
tional relaxation to their lowest vibrational level
(v0 ¼ 0). This process is extremely efficient with all the
excess vibrational energy being transferred to the
solvent molecules as heat in around 10
13–10
11 s.
In the case of excitation to S2, the process of internal
conversion to the upper vibrational levels of S1 (IC in
Figure 1) is rapid and effective. Thus luminescence in
solution will usually result from the lowest vibrational
level of the S1 excited state. This is not always the case
in gas-phase luminescence where collisional relaxa-
tion is far less likely. The fluorescence emission life-
time (t) is of similar magnitude to the mean lifetime of
the excited singlet state (10
10–10
7 s), the latter
being inversely proportional to the molar absorptivity.
As the probability of finding an excited state molecule
at time t after removal of the excitation source is
e
t/t, then the relationship between luminescence in-
tensity and the mean excited state lifetime is:
I ¼ I0 e
t=t ½1
where I is the luminescence intensity at time t and
I0 is the maximum luminescence intensity during
excitation. In practical terms the difference in t val-
ues for various analytes can provide extra selectivity
when using time-resolved luminescence spectroscopy.
Figure 1 also illustrates that the fluorescence emis-
sion spectrum is shifted to longer wavelengths com-
pared to that of the absorption (excitation) spectrum.
This is the result of vibrational relaxation both after
excitation and after fluorescence and is known as
Stokes’ shift. From the zeroth vibrational level of the
lowest excited singlet (n0 ¼ 0, S1) it is also possible for
radiationless deexcitation to convert all the energy to
heat in preference to light. This is termed internal
conversion, the quantum mechanical basis for which
is somewhat vague. The internal conversion from S1
308 LUMINESCENCE / Overview
to S0 for aliphatic compounds can be rationalized by
the overlapping of the upper vibrational levels of S0
with those of S1. In this case relaxation is rapid and
efficient and explains why aliphatic molecules rarely
luminesce. Internal conversion also occurs between
excited electronic states (as shown in Figure 1) due to
the overlapping vibrational levels of S1 and S2. At the
crossover point the potential energies of the two
excited states are equal and as the efficiency of vibra-
tional relaxation is far greater than emission of a
photon, internal conversion to the zeroth vibrational
level of S1 occurs rather than fluorescence from S2.
Therefore, fluorescence from anything but the S1
state is rare; the blue hydrocarbon azulene (isomeric
with naphthalene) is the most well-known exception.
In large molecules internal conversion may also re-
sult in predissociation (bond cleavage), when the
electron moves from an excited state to a high-
vibrational level of a lower electronic state. In this
situation the vibrational energy is sufficient to cause
cleavage of bonds in the unstable excited state.
Together with internal conversions, electronically
excited states can be deactivated by virtue of their
interactions with solvent molecules of other concom-
itant species present. Such pathways are termed ex-
ternal conversions. As a general rule, those environ-
mental parameters that lower the probability of
collisional deexcitation (such as lower temperature,
increased viscosity, and organized media) tend also to
enhance luminescence.
Whilst the probability of the direct population of
T1 from S0 by absorption is virtually zero, a kinet-
ically efficient pathway exists from the S1 state in a
number of molecules. The mechanism is known as
intersystem crossing and it can be considered as a
spin-dependent internal conversion. It should be
borne in mind that singlet–triplet transitions are ap-
proximately a million times less likely to occur than
singlet–singlet or triplet–triplet processes and also
that radiationless vibrational deexcitation occurs in
around 10
13 s. As the mechanism of intersystem
crossing relies upon vibrational coupling between S1
and T1, the time required for this spin forbidden
process can be estimated to be around 10
8–10
7 s.
This is of the same order as the lifetime of the ra-
diative transition and therefore intersystem crossing
competes with fluorescence. The probability of in-
tersystem crossing is enhanced when the energy dif-
ference between S1 and T1 is small and when the
lifetime of S1 is relatively long. Intersystem crossing
is also aided by the presence of heavy atoms (e.g.,
iodine and bromine) as substituents on either the
solute or the solvent molecules. This so-called
heavy-atom effect arises from increased spin/orbital
interactions and as such spin reversal becomes
more probable. After intersystem crossing, the
molecule rapidly undergoes internal conversion
(10
13–10
11 s) to the lowest vibrational level of T1.
In a similar manner to fluorescence, phosphores-
cence can only occur with a radiative deexcitation
from T1 to S0 and two factors limit the likelihood of
this event. Firstly, because the energy difference be-
tween S0 and T1 is smaller than that between S0 and
S1, the vibrational coupling between S0 and T1 may
be enhanced resulting in more efficient internal
conversion. Of more consequence, however, is the
relatively long intrinsic lifetime of the triplet excited-
state (10
4–102 s), which provides more than ample
opportunity for collisional deactivation. In fact, this
mechanism is predominant and explains why phos-
phorescence is rarely observed at room temperature
in simple solutions (2,3-butanedione and tris(2,20 -
bipyridyl)ruthenium(II) are examples). The relatively
long lifetime of T1 arises from the spin forbidden
nature of the triplet–singlet state transition; as a
consequence phosphorescence is often characterized
by an afterglow. This is not seen in fluorescence.
Clearly, radiationless processes are much more likely
to deexcite a triplet state compared to the ejection of
a photon, therefore, phosphorescence is most com-
monly observed from molecules that are either at
very low temperatures (often 77 K), in organized
media (micelles), or adsorbed onto solid surfaces.
Structural and Environmental Influences
on Photoluminescence
From the preceding section, it is evident that the
molecule’s structure and its chemical environment
determine whether or not it will luminesce, and to
what extent. In order to discuss these variables it is
useful to introduce quantum yield (f) or quantum
efficiency. For a luminescent process, f represents the
ratio of the number of molecules that emit to the
total number excited. For highly luminescent sub-
stances f will approach unity and for species that do
not luminesce appreciably, f will tend toward zero.
Thus the luminescence quantum yield for a particular
molecule in a specified environment will be related to
the relative rate constants of those pathways which
can deexcite the lowest excited state (S1 or T1). The
fluorescence quantum yield can therefore be ex-
pressed as follows:
kf
ff ¼ ½2
ðkf þ ki þ kr Þ
in which k represents the various rate constants
as designated by the subscripts, where f is fluores-
cence, i is intersystem crossing, and r is radiationless
energy loss, which includes internal and external

conversions plus predissociation or dissociation.
Thus, if kf cðki þ kr Þ then ff-1 and if kf {ðki þ
kr Þ then ff-0. The phosphorescence quantum yield
(fp) is dependent upon competition between the ra-
diationless routes from T1 to S0 and phosphorescence
emission. But fp also relies upon the rate of inter-
system crossing which competes with both fluores-
cence and radiationless deactivation from S1 and S0.
Thus fp is given by:
kp ki
fp ¼  ½3
kp þ k0r kf þ ki þ kr
where k0r represents the combined rate constant for
all radiationless pathways from T1 to S0. In the ma-
jority of situations kf and kp are related to molecular
structure with only minor dependence upon environ-
mental variables. The magnitude of ki can be affected
by both these parameters. Both kr and k0r have only
slight dependence upon molecular structure but are
markedly affected by the molecular environment.
Photoluminescence resulting from absorption of
wavelengths below 200 nm is not common since the
subsequent transitions from interaction with such
energetic photons are likely to result in deactivation
of the excited states via predissociation or dissocia-
tion. It is therefore not surprising that luminescence
due to s -s transitions are virtually nonexistent, in
fact most fluorescent or phosphorescent emissions
from organic molecules arise from p -p and some-
times p -n transitions depending upon which is less
energetic. Fluorescence is more commonly observed
from compounds where the S1-S0 transition corre-
sponds to a p -p emission rather than p -n, which
implies that the quantum efficiency resulting from
p; p states is far greater than that from n;p states.
This situation can be explained by consideration of
some spectroscopic parameters for each state (see
Table 1).
Because the lower molar absorptivity of the n-p
transitions translates to lower values of kf, the re-
lative importance (efficiency) of intersystem crossing
is in turn enhanced. The smaller energy differences
between S1 and T1 for n;p states also increases the
rate of intersystem crossing. Therefore, if S1 is an
Table 1 A comparison of n;p and p; p singlet states
n; p
Molar absorptivity (1 mol
1 cm
1) 10–103
Lifetime(s) 10
7–10
5
Energy difference between S1 Small
and T1
Rate constant for intersystem 4kf
crossing (ki)
LUMINESCENCE / Overview 309
n;p state, ki in eqn [2] becomes large with respect to
kf and as such ff tends toward zero. On the other
hand, when S1 is a p; p state, lifetimes are much
shorter (kiokf). There is also less vibrational level
overlap between S1 and T1 and consequently fluo-
rescence becomes more probable. In general, mole-
cules with low lying n;p states do not fluoresce but
may phosphoresce given a suitable environment. In
such a case emission would result from a p to n
transition, but phosphorescence is also observed
from p to p transitions.
Analytically usable fluorescence is most often
observed from compounds with aromatic function-
ality. Relatively few nonaromatic molecules exhibit
fluorescence and those that do often contain carbonyl
groups or are highly conjugated. Consistent with the
previous discussion, all these species possess low
energy p to p (S0 to S1) transitions. The majority of
unsubstituted aromatic hydrocarbons exhibit fluo-
rescence in liquid solution and sometimes phospho-
rescence under specific conditions. Increasing the
number of fused rings generally results in emission at
longer wavelengths; thus benzene and naphthalene
fluoresce in the UV whereas anthracene and naph-
thacene exhibit blue and green fluorescence, re-
spectively. Substitution on to the aromatic rings
causes shifts in the absorption wavelengths, which in
turn changes the emission spectra. More importantly,
substitution of particular species can drastically af-
fect the quantum efficiency. For example, large
(heavy) atoms increase the probability of intersys-
tem crossing to the triplet state and carboxylic acids
or carbonyl groups reduce the fluorescence quantum
yield since these moieties often possess low level n;p
transitions. Along with substitution, molecular
geometry also affects luminescence. This can be
illustrated by considering two structurally similar
compounds: fluorescein(1), which is intensely fluo-
rescent; and phenolphthalein(2), which is not fluo-
rescent. The only difference between the two
structures is the oxygen bridge present in fluoresce-
in, which imparts rigidity to the molecule. In phe-
nolphthalein electronic excitation can be lost
internally via vibration and rotation rather than
photon emission. In structurally rigid molecules,
energy dissipation via vibration and internal rota-
tion is far less efficient. Enhanced phosphorescence
can result from adsorption of the emitting species on
p; p to a solid surface to provide added rigidity. Such
103–105
molecular inflexibility is often used to rationalize the
10
10–10
7 observed increase in fluorescence intensity of organic
Often large complexing agents when chelated to a metal cation.
Thus, the complexes of Ca2 þ , Mg2 þ , Zn2 þ , and
okf Al3 þ with 8-quinolinol-5-sulfonic acid are much
more fluorescent than the free ligand molecule itself.
310 LUMINESCENCE / Overview
It is worth mentioning that while many metal ions
form rigid complexes with 8-quinolinol-5-sulfonic
acid, relatively few exhibit analytically useful fluo-
rescence. This is due to the internal heavy atom effect
and/or paramagnetism causing intersystem crossing,
which is manifested (in some instances) by increased
intensity of phosphorescence.
Together with molecular structure, environmental
parameters such as temperature, solvent type, visco-
sity, pH, and dissolved oxygen content can also affect
luminescence. As the solution temperature rises, it
follows that the number of collisions between the
excited state molecule and the solvent molecules will
increase, thus greatly improving the likelihood of
radiationless deexcitation to the ground state. There-
fore, ff for most compounds decreases with increa-
sing temperature. As mentioned earlier, the effect of
temperature upon fp is even more dramatic due to
the vastly greater lifetime of triplet states.
−O −O
O O O
COO− COO−
(1) (2)
By similar reasoning an increase in viscosity (via
organized media) will serve to limit collisional energy
transfer and thus enhance luminescence. The heavy
atom effect discussed earlier in relation to molecular
structure can be used to explain the depression of
fluorescence with solvents or solutes containing such
species. Carbon tetrabromide and ethyl iodide can be
used to limit fluorescence by promoting intersystem
crossing with a resultant enhancement of phospho-
rescence. The absorption of a photon causes a change
in electronic distribution and, therefore, molecular
geometry. This can often lead to a considerable dif-
ference in polarity between the ground and excited
states. If the excited state has increased polarity then
in polar solvents luminescence will be shifted to
longer wavelengths compared to that observed in a
nonpolar medium. This results from the increased
stabilization of the excited state relative to the
ground state. Solvent polarity can also affect the
type of luminescent phenomena observed. For ex-
ample, in cyclohexane, isoquinoline exhibits intense
phosphorescence (at 77 K), yet the same compound
in either water or ethanol gives only fluorescence. In
protic solvents the lone pair on the nitrogen (n-or-
bital) is solvated which lowers its energy relative to
the p orbital; as a consequence the p; p becomes
the lowest lying state and, as discussed earlier,
fluorescence is preferred. The reverse is the case in
cyclohexane, with the low lying n;p being prone to
intersystem crossing and then phosphorescence.
The control of pH in a solution containing a lu-
minescent analyte is also of great importance for
sensitive and reliable analysis. For aromatic com-
pounds with acidic or basic substituents, excitation
and emission wavelengths of the ionized and free
forms are likely to differ. In the case of fluorescence
from metal chelates, the pH must be controlled to
ensure that the conditional stability constant for
the complex is optimal for the particular analytical
situation.
Dissolved molecular oxygen can quench both flu-
orescence and phosphorescence albeit with different
efficiencies and mechanisms. The extent of oxygen
quenching on fluorescence is strongly dependent on
the fluorophore, whereas for phosphorescence the
intensity is always adversely affected. This can be
rationalized using the Stern–Volmer equation [4]
assuming no other quenching species are present.
fQ 1
¼ ½4
f0 ð1 þ kQ t½QÞ
where fQ and f0 are the luminescence quantum
yields in the presence and absence of the quencher,
respectively, [Q] is the molar concentration of the
quenching species and kQ is the rate constant for the
quenching interaction. Given that quenching is dif-
fusion limited (kQE1010 mol
1 l s
1) and that the
concentration of oxygen in water at atmospheric
pressure is B10
3 mol l
1 then eqn [4] becomes:
fQ 1
¼ ½5
f0 ð1 þ 107 tÞ
Since phosphorescence lifetimes in the absence of
oxygen are in the range 10
4–102 s, the ratio of the
quantum yields (from eqn [5]) will be extremely
small for most phosphorescent species. However, as
fluorescent lifetimes in the absence of oxygen vary
from 10
10 to 10
7 s, the diffusion controlled
oxygen quenching of singlet states will be most ef-
ficient for the longer-lived species. Numerous mech-
anisms have been proposed for the quenching of
fluorescence by molecular oxygen. The most likely
pathway involves enhancement of intersystem cross-
ing by the triplet oxygen, which can be summarized
as follows:
1
X þ3 O2 -ðXþ þ O
3  3
2 Þ- X þ O2
The mechanism of intersystem crossing (1X to 3X )
is thought to occur via a transient charge-transfer
species between the excited fluorophore (X ) and
molecular oxygen. Although molecular oxygen
promotes intersystem crossing, it does not enhance
 LUMINESCENCE / Overview 311

phosphorescence. As discussed earlier, the long life- Entrance

b1 b2
time of the radiative transition precludes phospho- slit
rescence being observed in simple solution due to
quenching by collisional deactivation. The mecha- Sample cell
nism for the quenching of triplet states in solution by
Excitation
molecular oxygen (or other triplets) is at best spec- radiation P0 P ′0 P′
ulative, possibly involving triplet–triplet annihila- (
ex)
tion. The ability of molecular oxygen to efficiently I′
b3
quench photoluminescence can, however, be exploit-
ed for the sensitive determination of oxygen in liq-
uids and solids. I
Exit
Paramagnetic transition metal ions may also slit
quench solution luminescence by increasing the rate
of intersystem crossing. However, there are numer-
ous exceptions to this simple mechanism including Photoluminescent
manganese(II) ions, which are generally poor quenc- emission (
em)
hers of luminescence but are highly paramagnetic.
Figure 2 A schematic representation of the sample compart-
Diamagnetic main group metal ions are usually in-
ment of a photoluminescent spectrometer.
efficient quenching agents. It is therefore clear that
other processes may be involved in the interaction of
where eem is the molar absorptivity at lem. By rear-
metal ions with luminescent species. Excited state
ranging eqns [6]–[9] the following relationship
complex formation and energy transfer are both
between P0 and I can be obtained:
likely in some instances. From a practical analytical
point of view, the quenching of luminescence by any I ¼ fP0 10
eex b1 C ð1
10
eex b2 C Þ10
eem b3 C ½10
concomitant species in real samples must be inves- At low analyte concentrations I is proportional to
tigated during method development and either C as absorption is small. Since the absorbance in-
removed or compensated for. creases faster than emission at high analyte concen-
The Relationship between Photoluminescence trations the resultant I value reaches a maximum and
Intensity and Analyte Concentration then decreases as seen in Figure 3. This is termed self-
absorption and it occurs when lem overlaps the
The relationship between emission intensity and absorption band. Self-quenching also decreases the
analyte concentration can be derived using the Lam- luminescence intensity at high concentrations as it
bert–Beer law in conjunction with the schematic of a results from a radiationless energy transfer between
spectroluminometer in Figure 2. two excited molecules and the solvent in a similar
If the incident radiation (lex) has the power P0 and fashion to external conversion. Under certain condi-
a portion of this is absorbed over b1 then the radiant tions self-absorption and self-quenching may result
power striking the central region of the sample (P00 ) in a maximum in the calibration function. Equation
is given by: 0 [10] can be simplified at low analyte concentrations;
P0 ¼ P0 10
eex b1 C ½6 absorbance will be negligible and the three exponents
where eex is the molar absorptivity at lex. It follows will become very small. Therefore, we can replace
that the radiant power of the exciting beam after the terms 10
eex b1 C and 10
eem b3 C by unity. Clearly,
traversing b2 (P0 ) is: the term 10
eex b2 C cannot be treated in this way or
0 0
eqn [10] would become zero:
P ¼ P0 10
eex b2 C ½7
I ¼ fP0 ð1
10
eex b2 C Þ ½11
The luminescence intensity (I0 ) is proportional to
both the amount of light absorbed and the quantum The term in eqn [11] can subsequently be expanded
efficiency (f) such that: as a MacLaurin series to give:
"
0 0
I ¼ fðP0
P Þ
0
½8 ðeex b2 C ln 10Þ2
I ¼ fP0 eex b2 C ln 10

2!
A fraction of the emitted radiation will be ab- #
sorbed over the pathlength b3 and this can be ðeex b2 C ln 10Þ3 ðeex b2 C ln 10Þn
þ ?þ ½12
expressed in terms of the observed luminescence in- 3! n!
tensity (I):
0
When the solution absorbance is small (eexb2
I ¼ I 10
eem b3 C ½9 Co0.05) it is a good approximation to neglect all
312 LUMINESCENCE / Overview
200
Fluorescence intensity (mV)
150
100
50
0.0 0.1 0.2 0.3 0.4 0.5
Ru(bipy)3 concentration (mmol l −1)
2+
Figure 3 Photoluminescence calibration for an aqueous solu-
tion of tris(2,20 -bipyridyl)ruthenium(II) chloride hexahydrate,
lex ¼ 457 nm, lem ¼ 609 nm.
but the first term in the series:
I ¼ fP0 eex b2 C ln 10 ½13
Therefore, when the absorbance is small (at constant
P0 and under a defined chemical environment) the
observed emission intensity is directly proportional
to analyte concentration:
I ¼ KC ½14
In most analytical applications the above condi-
tions are met; and although the linear dependence of
I upon P0 is not infinite, it provides a significant
advantage for photoluminescence over absorption
spectrophotometry.
Excitation and Emission Spectra
Figure 4 shows both the emission (A) and excitation
(B) spectra of anthracene in ethanol (c. 1 mg ml
1).
The former was recorded with a fixed excitation
wavelength of 340 nm and the latter was monitored
at an emission wavelength set to 379 nm. The four
peaks in Figure 4A are transitions from the zeroth
vibrational level of S1 to the excited vibrational levels
of S0. Conversely, the four peaks in Figure 4B cor-
respond to transitions between the zeroth vibrational
level of S0 and the various vibrational levels of S1.
The approximate ‘mirror image’ appearance of the
two spectra occurs due to the similarities in the
energies of the vibrational levels in S0 and S1.
The difference in wavelength between the excita-
tion and emission spectra is a characteristic of photo-
luminescent molecules and is known as the Stokes’
shift, which can be quantified into wavenumbers by
Relative fluorescence intensity
(A)
300 350 400 450 500
(B) Wavelength (nm)
Figure 4 Fluorescence spectra of anthracene (1 mg ml
1) in
ethanol. The emission spectrum (A) was obtained with
lex ¼ 340 nm and the excitation spectrum (B) was obtained with
lem ¼ 379 nm.
the following relationship:


0 1 1
Stokes shift ¼ 10 7

½15
lex lem
where lex and lem are the corrected wavelengths of
the maximum excitation and emission, respectively
(in nanometers). In order to record corrected excita-
tion and emission spectra the wavelength dependence
of the source, monochromators, and photomultiplier
response must be identified and accounted for. The
presence of the extraneous peak in the emission
spectrum can be attributed to Rayleigh scattering of
the excitation radiation. This is most commonly
observed at either the excitation wavelength or twice
this value due to second order diffraction from the
grating in the emission spectrometer. Anthracene also
exhibits phosphorescence in ethanol at 77 K with the
wavelength of maximum emission being 462 nm.
This is consistent with the lower energy of the lowest
excited triplet state.
Chemiluminescence
All chemical reactions are accompanied by energy
changes. Any excess energy is usually dissipated by
collision in around 10
12 s. Chemiluminescence is
commonly observed at wavelengths from the near
ultraviolet to the near infrared (which correspond to

excess chemical energy in the range from 340 to
130 kJ mol
1, respectively). Whilst these amounts of
excess energy are achievable with certain reactions
(often redox), the generation of chemiluminescence
depends upon the suitable molecular structure of in-
termediates or products that will facilitate the
conversion of chemical potential to electronic exci-
tation.
After chemical generation of the excited state has
occurred, all of the environmental factors that influ-
ence photoluminescence are equally valid for chemi-
luminescence. It is generally accepted that most
chemiluminescence reactions can be thought of as
chemically induced fluorescence based upon the rar-
ity of phosphorescence occurring in simple solution.
Two noteworthy exceptions are the reduction of (1)
tris(2,20 -bipyridyl)ruthenium(III) and (2) acidic
potassium permanganate in the presence of certain
polyphosphates. The emission in these cases appears
to originate from very short-lived triplets. Many
chemiluminescent reactions can produce light for
several minutes or even longer. It should be empha-
sized that this is indicative only of the reaction ki-
netics, which produce the emitting species, and not
the lifetime of the excited state.
Chemiluminescence can be rather simply classified
as either direct or indirect. The former can be
thought of as follows:
A þ B-C þ D
C -C þ hn
where A and B are reactants employed to produce
either a product or intermediate in an electronically
excited state (C ) that returns to its ground state by
ejection of a photon (hn).
Indirect chemiluminescence has been used ex-
tensively for analysis and in the so-called chemical
light sources; the most well known of these reactions
involve oxidation of certain diaryl oxalates and ox-
amides. Instead of C returning to the ground state
by photon ejection (as in the above reaction scheme),
it can undergo energy transfer with a suitable fluor-
ophore, which in turn may then exhibit its charac-
teristic fluorescence emission:
C þ fluorophore-½fluorophore þProducts
½fluorophore -fluorophore þ hn
0
The analytical utility of indirect chemilumines-
cence depends upon little or no emission resulting
from C in the region of maximum emission from the
electronically excited fluorophore together with effi-
cient transfer of excitation energy.
LUMINESCENCE / Overview 313
Bioluminescence
The emission of light from fireflies has enchanted
observers for millennia. However, it was not until
comparatively recently (1947) that adenosine tripho-
sphate (ATP) was identified as being the key compo-
nent in the enzymatically controlled luciferin
luminescence. The analytical utility of firefly biolu-
minescence was first demonstrated in 1952. Since
that time the luciferin–luciferase reaction has been
extensively employed for analysis in a wide variety of
scientific disciplines.
While there are numerous species of firefly having
similar biochemical pathways for the generation of
light, the proposed mechanism for the luciferin–
luciferase reaction is based upon the system of Photi-
nus pyralis. The oxidation of D-luciferin (3) by
oxygen, which is catalyzed by the enzyme luciferase,
is thought to proceed by the sequence outlined
below:
Mg2þ
E þ LH2 þ ATP " E-LH2 -AMP þ PP
E-LH2 -AMP þ O2 -E þ L ¼ O þ CO2 þ AMP
where E is the enzyme luciferase, L is luciferin
(3), AMP is adenosine monophosphate, PP is
pyrophosphate and L ¼ O is oxyluciferin (4).
N N
COOH N N O
H
S S H S S
HO H HO
(3) (4)
In the initial step, the D-luciferin is activated via
interaction with Mg-ATP to give the enzyme bond D-
luciferyl adenylate and pyrophosphate. As shown,
this reaction is reversible and therefore pyrophos-
phate can impede the forward reaction. Generally,
only low levels of pyrophosphate are formed from
the concentrations of ATP and luciferase present un-
der normal reaction conditions. The second step is
an oxidative decarboxylation which produces ox-
yluciferin (4) in an electronically excited state. The
excited oxyluciferin returns to the ground state by
the ejection of a photon (lem ¼ 562 nm) with an im-
pressive overall quantum yield of B0.9 (at 251C and
pH 7.8). The light intensity is directly proportional
to the ATP concentration provided that the level of
luciferin remains constant.
There are numerous other bioluminescent systems
of analytical importance, which have been isolated
from various natural sources. A more detailed
314 LUMINESCENCE / Overview

Table 2 Selected analytica applications of other luminescent phenomena

Class Analyte Comments Reference

Sonoluminescence
Thermoluminescence
Candoluminescence
Radioluminescence
Ionoluminescence
Dissolved oxygen
Tetragonal zirconia in
alumina–zirconia powders
Lead and tellurium
Phosphorus
Chemical states of iron in a
plagioclase sample
No interference from anions
present in natural waters
Characterized by a peak at

351C.
Matrix coated on a rod before
placing in a hydrogen flame
Photoluminescent technique
with a light source based on
radioluminescence
External proton beam induced
peaks at 553 nm (Fe2 þ ) and
682 nm (Fe3 þ )
Yan L, Ruo F, and Zhaohua C
(1995). Water Research 29:
2014
Salle C et al. (2003). Journal of
European Ceramic Society
23: 667
Kassir ZM and Taher MB
(1985). Analyst 110: 1223
Leach AM, Burden DL, and
Hieftje GM (1999).
Analytical Chimica Acta 402:
267
Sha Y et al. (2002). Nuclear
Instruments and Methods in
Physical Research Section
B 189: 408

discussion of these systems is given elsewhere in this See also: Bioluminescence. Chemiluminescence:
encyclopedia. Overview. Fluorescence: Overview. Phosphorescence:
Principles and Instrumentation.

Other Types of Luminescence

Other classes of luminescence have been used in an-
alytical applications; some examples are provided in
Table 2. Thermally stimulated luminescence (or
thermoluminescence) is the emission arising during
mild heating of a solid material, often after it has
been subjected to ionizing radiation. Thermolumi-
nescence has been extensively employed for dating
and dosimetry, including (for example) the detection
of irradiated foods. Some incandescent solids emit at
much shorter wavelengths than expected due to
‘candoluminescence’, which has been exploited for
the detection of metals such as manganese, antimony,
cerium, europium, terbium, lead, and bismuth.
Radioluminescence – induced by gamma- or X-
rays – has also been exploited for dating. Radiolu-
minescent light sources have been designed for spec-
trometric studies; the radioisotope and scintillation
medium allows independent selection of the spectral
and temporal characteristics. Sonoluminescence, the
emission observed when particular solutions are ex-
posed to ultrasonic waves, has the potential for an-
alytical applications involving the detection of
medium-to-high concentrations (over 10 g dm
3) of
elements that have an ionization energy below
7.65 eV and a boiling point below 27001C.
Triboluminescence (arising from solids during
structural rearrangements such as crushing) has lim-
ited applicability in analytical chemistry, but has
been examined as a tool for clinical diagnosis with
blood samples. The use of triboluminescent materials
to detect damage in composite structures during im-
pact has been suggested.
Further Reading
Baeyens WRG, de Keukeliere D, and Korkidis K (eds.)
(1991) Luminescence Techniques in Chemical and Bio-
chemical Analysis. New York: Dekker.
Burr JG (ed.) (1985) Chemi- and Bioluminescence. New
York: Dekker.
Campbell AK (1988) Chemiluminescence Principles and
Applications in Biology and Medicine. Chichester: Ellis
Horwood.
Garcı́a-Campaña AN and Baeyens WRG (eds.) (2001)
Chemiluminescence in Analytical Chemistry. New York:
Dekker.
Guilbault GG (ed.) (1990) Practical Fluorescence, 2nd edn.
New York: Dekker.
Harris DC (1991) Quantitative Chemical Analysis, 3rd
edn., pp. 530–532. New York: W.H. Freeman.
Harvey EN (1957) A History of Luminescence from the
Earliest Times until 1900. Philadelphia: The American
Philosophical Society.
Hercules DM (ed.) (1966) Fluorescence and Phosphores-
cence Analysis Principles and Application. New York:
Interscience.
Kricka LJ and Carter TJN (eds.) (1982) Clinical and Bio-
chemical Luminescence. New York: Dekker.
Lakowicz JR (1999) Principles of Fluorescence Spectro-
scopy, 2nd edn. New York: Kluwer Academic/Plenum.
Schulman SG (ed.) (1985) Molecular Luminescence Spec-
troscopy. Methods and Applications, part 1. New York:
Wiley-Interscience.
Schulman SG (ed.) (1988) Molecular Luminescence Spec-
troscopy. Methods and Applications, part 2. New York:
Wiley-Interscience.
Schulman SG (ed.) (1993) Molecular Luminescence Spec-
troscopy. Methods and Applications, part 3. New York:
Wiley-Interscience.

Skoog DA, Holler FJ, and Nieman TA (1998) Principles of
Instrumental Analysis, 5th edn., pp. 355–379. Fort
Worth: Saunders College Publishing.
Valeur B (2002) Molecular Fluorescence Principles and
Applications. Weinheim: Wiley-VCH.
Solid Phase
L F Capitán-Vallvey, University of Granada, Granada, Spain
& 2005, Elsevier Ltd. All Rights Reserved.
Introduction
Luminescence – fluorescence, phosphorescence, and
even chemiluminescence – may be emitted from the
surface of powdered solid supports with small-sized
particles or from membranes coming from com-
pounds immobilized by physical or chemical proce-
dures when exposed to by external radiation. The
selectivity, sensitivity, speed, flexibility, and simplic-
ity of solid-phase luminescence spectrometry (SPLS)
make it a good analytical tool, especially in trace
analysis. Because of its ease in handling, the use of
SPLS has increased in different fields of interest and
many different formats have emerged.
The reasons for the increase in the use of SPLS lie
in its multidimensional character (spectral, lifetime,
polarization, and other measurements) as well as in
the need for a system that shows a large number of
reactions and processes very efficiently. The advan-
tages of SPLS include its sensitivity, low cost, ease in
performance, versatility, and that it offers subnano-
meter spatial resolution with submicrometer vis-
ualization and submillisecond temporal resolution.
The reasons for the widespread use of SPLS lumi-
nescence are varied and generally make use of: im-
provements in the photophysical emission process,
convenience of use, possibility of preconcentration,
and use of a solid phase as a carrier for reactions or
as a matrix for depositing or protecting reagents.
Referring here only to analytical purposes, SPLS is
used for detection or determination of intrinsically
fluorescent or phosphorescent compounds, for non-
luminescent compounds that are capable of showing
luminescence when they are derivatized, for non-
luminescent, nonderivatized compounds that are ca-
pable of modifying the luminescent properties of a
probe, i.e., via quenching or solvatochromic effect,
and for compounds that interact via a binding part-
ner and indicate this reaction with a luminescent
label.
LUMINESCENCE / Solid Phase 315
van Wandruszka K (1992) Luminescence of micellar
solutions. Critical Reviews in Analytical Chemistry 23:
187–215.
Wolfbeis OS (ed.) (1993) Fluorescence Spectroscopy. New
Methods and Applications. Heidelberg: Springer.
The different ways in which luminescence in solid
phases is measured are: as a simple support, i.e., in
intrinsically fluorescent compounds; as a support
that makes a luminescent process possible in certain
chemical conditions, i.e., phosphorescence; as a
phase for preconcentrating the analyte or a derivative
from a diluted solution due to the favorable distri-
bution constant, i.e., polycyclic aromatic hydrocar-
bons (PAHs) on paper or C18-silica; as a phase that
contains one or more immobilized reagents in a
monolayer or multilayer format, enabling the reac-
tion and retention of the analyte as well as different
processes such as separation or others, i.e., test strips;
as a phase that contains a compound that modifies its
luminescent characteristics upon contact with the
analyte, i.e., oxygen acting on a metallophorphyrin
embedded in a polymer.
Principles
Fluorescence arises from transitions that occur be-
tween singlet states and is not significantly modified
upon being emitted from a solid substrate with re-
spect to the solution, although in general the quan-
tum yield is higher than in solution, while in the case
of phosphorescence there are striking differences in
quantum yield between solid and solution. Phospho-
rescence generally involves an intersystem crossing to
a triplet state, and a subsequent radiative transition
that is rather slow. In this case, nonradiative relax-
ation is the usual deactivation process of the long
lifetime excited triplet state, due to intermolecular
collisional quenching processes and intramolecular
vibrational–rotational relaxation. Fixing a potential-
ly phosphorescent analyte on a solid phase hinders its
motion in this rigid environment and restricts radi-
ationless deactivation, protecting the triplet state and
increasing the quantum yield. There are two main
solid-phase immobilization techniques: physical trap-
ping in the rigid glass formed at low temperature and
retention on a solid phase at room temperature. We
are concerned with this second technique.
While in solution luminescence components are
dissolved, in SPLS the electromagnetic radiation that