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Chapter 10 Spectroscopic Methods of Analysis 423

10F Spectroscopy Based on Emission

An analyte in an excited state possesses an energy, E2, that is greater than that when
it is in a lower energy state, E1. When the analyte returns, or relaxes to a lower en-
ergy state the excess energy, ∆E,
∆E = E2 – E1
must be released. Figure 10.5 shows a simplified picture of this process.
The lifetime of an analyte in the excited state, A*, is short; typically 10–5–10–9 s lifetime
for electronic excited states and 10–15 s for vibrational excited states. Relaxation oc- The length of time that an analyte stays
curs through collisions between A* and other species in the sample, by photochemi- in an excited state before returning to a
lower-energy state.
cal reactions, and by the emission of photons. In the first process, which is called vi-
brational deactivation, or nonradiative relaxation, the excess energy is released as relaxation
heat; thus Any process by which an analyte returns
to a lower-energy state from a higher-
A* → A + heat energy state.
Relaxation by a photochemical reaction may involve a decomposition reaction in
which A* splits apart
A* → X + Y
or a reaction between A* and another species
A* + Z → X + Y
In either case the excess energy is used up in the chemical reaction or released as
heat.
In the third mechanism excess energy is released as a photon of electromagnetic
radiation.
A* → A + hν
The release of a photon following thermal excitation is called emission, and that fol-
lowing the absorption of a photon is called photoluminescence. In chemilumines-
cence and bioluminescence, excitation results from a chemical or biochemical reac-
tion, respectively. Spectroscopic methods based on photoluminescence are the
subject of Section 10G, and atomic emission is covered in Section 10H.

10G Molecular Photoluminescence Spectroscopy

Photoluminescence is divided into two categories: fluorescence and phosphores-
cence. Absorption of an ultraviolet or visible photon promotes a valence electron
from its ground state to an excited state with conservation of the electron’s spin. For
example, a pair of electrons occupying the same electronic ground state have oppo-
site spins (Figure 10.42a) and are said to be in a singlet spin state. Absorbing a pho- singlet excited state
ton promotes one of the electrons to a singlet excited state (Figure 10.42b). Emis- An excited state in which all electron
sion of a photon from a singlet excited state to a singlet ground state, or between spins are paired.
any two energy levels with the same spin, is called fluorescence. The probability of a fluorescence
fluorescent transition is very high, and the average lifetime of the electron in the ex- Emission of a photon when the analyte
cited state is only 10–5–10–8 s. Fluorescence, therefore, decays rapidly after the exci- returns to a lower-energy state with the
tation source is removed. In some cases an electron in a singlet excited state is trans- same spin as the higher-energy state.
formed to a triplet excited state (Figure 10.42c) in which its spin is no longer triplet excited state
paired with that of the ground state. Emission between a triplet excited state and a An excited state in which unpaired
singlet ground state, or between any two energy levels that differ in their respective electron spins occur.
1400-CH10 9/8/99 4:19 PM Page 424
424 Modern Analytical Chemistry
Singlet
ground state
(a)
Figure 10.42
Difference between singlet and triplet states.
phosphorescence
Emission of a photon when the analyte
returns to a lower-energy state with the
opposite spin as the higher-energy state.
vibrational relaxation
A form of radiationless relaxation in
which an analyte moves from a higher
vibrational energy level to a lower
vibrational energy level in the same
electronic level.
Singlet Triplet
excited state excited state
(b) (c)
spin states, is called phosphorescence. Because the average lifetime for phosphores-
cence ranges from 10–4 to 104 s, phosphorescence may continue for some time after
removing the excitation source.
The use of molecular fluorescence for qualitative analysis and semiquantitative
analysis can be traced to the early to mid-1800s, with more accurate quantitative
methods appearing in the 1920s. Instrumentation for fluorescence spectroscopy
using filters and monochromators for wavelength selection appeared in, respec-
tively, the 1930s and 1950s. Although the discovery of phosphorescence preceded
that of fluorescence by almost 200 years, qualitative and quantitative applications of
molecular phosphorescence did not receive much attention until after the develop-
ment of fluorescence instrumentation.
10G.1 Molecular Fluorescence and Phosphorescence Spectra
To appreciate the origin of molecular fluorescence and phosphorescence, we must
consider what happens to a molecule following the absorption of a photon. Let’s as-
sume that the molecule initially occupies the lowest vibrational energy level of its
electronic ground state. The ground state, which is shown in Figure 10.43, is a sin-
glet state labeled S0. Absorption of a photon of correct energy excites the molecule
to one of several vibrational energy levels in the first excited electronic state, S1, or
the second electronic excited state, S2, both of which are singlet states. Relaxation to
the ground state from these excited states occurs by a number of mechanisms that
are either radiationless, in that no photons are emitted, or involve the emission of a
photon. These relaxation mechanisms are shown in Figure 10.43. The most likely
pathway by which a molecule relaxes back to its ground state is that which gives the
shortest lifetime for the excited state.
Radiationless Deactivation One form of radiationless deactivation is vibra-
tional relaxation, in which a molecule in an excited vibrational energy level loses
energy as it moves to a lower vibrational energy level in the same electronic state.
Vibrational relaxation is very rapid, with the molecule’s average lifetime in an
excited vibrational energy level being 10–12 s or less. As a consequence, molecules
that are excited to different vibrational energy levels of the same excited elec-
tronic state quickly return to the lowest vibrational energy level of this excited
state.
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Chapter 10 Spectroscopic Methods of Analysis 425

S2
vr
vr
vr S1
vr vr
ic vr
vr T1
vr isc
vr
vr
ic vr
and
ec

ec

vr vr
Figure 10.43
Energy level diagram for a molecule showing
S0 vr vr pathways for deactivation of an excited
vr vr state: vr is vibrational relaxation; ic is internal
vr vr conversion; ec is external conversion, and isc
is intersystem crossing. The lowest
vibrational energy level for each electronic
Fluorescence Phosphorescence state is indicated by the thicker line.

Another form of radiationless relaxation is internal conversion, in which a internal conversion

molecule in the ground vibrational level of an excited electronic state passes directly A form of radiationless relaxation in
which the analyte moves from a higher
into a high vibrational energy level of a lower energy electronic state of the same
electronic energy level to a lower
spin state. By a combination of internal conversions and vibrational relaxations, a electronic energy level.
molecule in an excited electronic state may return to the ground electronic state
without emitting a photon. A related form of radiationless relaxation is external external conversion
A form of radiationless relaxation in
conversion in which excess energy is transferred to the solvent or another compo-
which energy is transferred to the solvent
nent in the sample matrix. or sample matrix.
A final form of radiationless relaxation is an intersystem crossing in which a
molecule in the ground vibrational energy level of an excited electronic state passes intersystem crossing
A form of radiationless relaxation in
into a high vibrational energy level of a lower energy electronic energy state with a which the analyte moves from a higher
different spin state. For example, an intersystem crossing is shown in Figure 10.43 electronic energy level to a lower
between a singlet excited state, S1, and a triplet excited state, T1. electronic energy level with a different
spin state.
Fluorescence Fluorescence occurs when a molecule in the lowest vibrational en-
ergy level of an excited electronic state returns to a lower energy electronic state by
emitting a photon. Since molecules return to their ground state by the fastest mech-
anism, fluorescence is only observed if it is a more efficient means of relaxation than
the combination of internal conversion and vibrational relaxation. A quantitative
expression of the efficiency of fluorescence is the fluorescent quantum yield, Φf, quantum yield
which is the fraction of excited molecules returning to the ground state by fluores- The fraction of absorbed photons that
cence. Quantum yields range from 1, when every molecule in an excited state un- produce a desired event, such as
fluorescence or phosphorescence (Φ).
dergoes fluorescence, to 0 when fluorescence does not occur.
The intensity of fluorescence, If, is proportional to the amount of the radiation
from the excitation source that is absorbed and the quantum yield for fluorescence
If = kΦf(P0 – PT) 10.29
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426 Modern Analytical Chemistry

where k is a constant accounting for the efficiency of collecting and detecting the
fluorescent emission. From Beer’s law we know that
PT
= 10 −εbC 10.30
P0
where C is the concentration of the fluorescing species. Solving equation 10.30 for
PT and substituting into equation 10.29 gives, after simplifying
If = kΦfP0(1 – 10–εbC) 10.31
For low concentrations of the fluorescing species, where εbC is less than 0.01, this
equation simplifies to
If = 2.303kΦfP0εbC 10.32
The intensity of fluorescence therefore, increases with an increase in quantum effi-
ciency, incident power of the excitation source, and the molar absorptivity and con-
centration of the fluorescing species.
Fluorescence is generally observed with molecules where the lowest energy ab-
sorption is a π → π* transition, although some n → π* transitions show weak fluo-
rescence. Most unsubstituted, nonheterocyclic aromatic compounds show favorable
fluorescence quantum yields, although substitution to the aromatic ring can have a
significant effect on Φf. For example, the presence of an electron-withdrawing
group, such as —NO2, decreases Φf, whereas adding an electron-donating group,
such as —OH, increases Φf. Fluorescence also increases for aromatic ring systems
and for aromatic molecules with rigid planar structures.
A molecule’s fluorescence quantum yield is also influenced by external vari-
ables such as temperature and solvent. Increasing temperature generally decreases
Φf because more frequent collisions between the molecule and the solvent increases
external conversion. Decreasing the solvent’s viscosity decreases Φf for similar rea-
sons. For an analyte with acidic or basic functional groups, a change in pH may
change the analyte’s structure and, therefore, its fluorescent properties. Changes in
both the wavelength and intensity of fluorescence may be affected.
As shown in Figure 10.43, fluorescence may return the molecule to any of sev-
eral vibrational energy levels in the ground electronic state. Fluorescence, therefore,
occurs over a range of wavelengths. Because the change in energy for fluorescent
emission is generally less than that for absorption, a molecule’s fluorescence spec-
trum is shifted to higher wavelengths than its absorption spectrum.

Phosphorescence A molecule in the lowest vibrational energy level of an excited

triplet electronic state normally relaxes to the ground state by an intersystem cross-
ing to a singlet state or by external conversion. Phosphorescence is observed when
relaxation occurs by the emission of a photon. As shown in Figure 10.43, phospho-
rescence occurs over a range of wavelengths, all of which are at a lower energy than
the molecule’s absorption band. The intensity of phosphorescence, Ip, is given by an
equation similar to equation 10.32 for fluorescence
Ip = 2.303kΦpP0εbC 10.33
where Φp is the quantum yield for phosphorescence.
Phosphorescence is most favorable for molecules that have n → π* transitions,
which have a higher probability for an intersystem crossing than do π → π* transi-
tions. For example, phosphorescence is observed with aromatic molecules contain-
ing carbonyl groups or heteroatoms. Aromatic compounds containing halide atoms
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Chapter 10 Spectroscopic Methods of Analysis 427

also have a higher efficiency for phosphorescence. In general, an increase in phos-

phorescence corresponds to a decrease in fluorescence.
Since the average lifetime for phosphorescence is very long, ranging from 10–4
to 104 s, the quantum yield for phosphorescence is usually quite small. An improve-
ment in Φp is realized by decreasing the efficiency of external conversion. This may
be accomplished in several ways, including lowering the temperature, using a more
viscous solvent, depositing the sample on a solid substrate, or trapping the molecule
in solution.

Excitation Versus Emission Spectra Photoluminescence spectra are recorded by

measuring the intensity of emitted radiation as a function of either the excitation
wavelength or the emission wavelength. An excitation spectrum is obtained by excitation spectrum
monitoring emission at a fixed wavelength while varying the excitation wavelength. A fluorescence or phosphorescence
Figure 10.44 shows the excitation spectrum for the hypothetical system described by spectrum in which the emission intensity
the energy level diagram in Figure 10.43. When corrected for variations in source at a fixed wavelength is measured as a
intensity and detector response, a sample’s excitation spectrum is nearly identical to function of the wavelength used for
excitation.
its absorbance spectrum. The excitation spectrum provides a convenient means for
selecting the best excitation wavelength for a quantitative or qualitative analysis.
In an emission spectrum a fixed wavelength is used to excite the mol-
ecules, and the intensity of emitted radiation is monitored as a function of
Fluorescent
wavelength. Although a molecule has only a single excitation spectrum, it emission
has two emission spectra, one for fluorescence and one for phosphores- S0 S1 spectrum
cence. The corresponding emission spectra for the hypothetical system in Phosphorescent
Figure 10.43 are shown in Figure 10.44. emission

Emission intensity
Excitation
spectrum
spectrum

10G.2 Instrumentation S0 S2
The basic design of instrumentation for monitoring molecular fluores-
cence and molecular phosphorescence is similar to that found for other
spectroscopies. The most significant differences are discussed in the fol-
lowing sections.
Wavelength
Molecular Fluorescence A typical instrumental block diagram for molec-
Figure 10.44
ular fluorescence is shown in Figure 10.45. In contrast to instruments for Example of molecular excitation and
absorption spectroscopy, the optical paths for the source and detector are emission spectra.
usually positioned at an angle of 90°.

Chopper
Excitation
Source wavelength Sample
selector

Emission
wavelength
selector

Signal Figure 10.45

Detector
processor Block diagram for molecular fluorescence
spectrometer.
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428 Modern Analytical Chemistry

fluorometer
An instrument for measuring
fluorescence that uses filters to select the
excitation and emission wavelengths.
spectrofluorometer
An instrument for measuring
fluorescence that uses a
monochromator to select the excitation
and emission wavelengths.
Two basic instrumental designs are used for measuring molecular fluorescence.
In a fluorometer the excitation and emission wavelengths are selected with absorp-
tion or interference filters. The excitation source for a fluorometer is usually a low-
pressure mercury vapor lamp that provides intense emission lines distributed
throughout the ultraviolet and visible region (254, 312, 365, 405, 436, 546, 577, 691,
and 773 nm). When a monochromator is used to select the excitation and emission
wavelengths, the instrument is called a spectrofluorometer. With a monochroma-
tor, the excitation source is usually a high-pressure Xe arc lamp, which has a contin-
uum emission spectrum. Either instrumental design is appropriate for quantitative
work, although only a spectrofluorometer can be used to record an excitation or
emission spectrum.
The sample cells for molecular fluorescence are similar to those for optical
molecular absorption. Remote sensing with fiber-optic probes (see Figure 10.30)
also can be adapted for use with either a fluorometer or spectrofluorometer. An an-
alyte that is fluorescent can be monitored directly. For analytes that are not fluores-
cent, a suitable fluorescent probe molecule can be incorporated into the tip of the
fiber-optic probe. The analyte’s reaction with the probe molecule leads to an in-
crease or decrease in fluorescence.

Molecular Phosphorescence Instrumentation for molecular phosphorescence

must discriminate between phosphorescence and fluorescence. Since the lifetime
for fluorescence is much shorter than that for phosphorescence, discrimination is
easily achieved by incorporating a delay between exciting and measuring phospho-
rescent emission. A typical instrumental design is shown in Figure 10.46. As shown

Chopper Chopper

Excitation Emission
Source wavelength Sample wavelength
selector selector

Detector

Signal
processor

(a) Excitation
Chopper Chopper

From To
Sample detector
source

(b) Emission
Chopper Chopper

From To
Sample detector
Figure 10.46 source
Block diagram for molecular
phosphorescence spectrometer with inset
showing how choppers are used to isolate
excitation and emission.
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Chapter 10 Spectroscopic Methods of Analysis 429

in the inset, the two choppers are rotated out of phase, such that fluorescent emis-
sion is blocked from the detector when the excitation source is focused on the sam-
ple, and the excitation source is blocked from the sample when measuring the phos-
phorescent emission.
Because phosphorescence is such a slow process, provision must be made to
prevent deactivation of the excited state by external conversion. Traditionally, this
has been accomplished by dissolving the sample in a suitable organic solvent, usu-
ally a mixture of ethanol, isopentane, and diethyl ether. The resulting solution is
frozen at liquid-N2 temperatures, forming an optically clear solid. The solid matrix
minimizes external conversion due to collisions between the analyte and the sol-
vent. External conversion also is minimized by immobilizing the sample on a solid
substrate, allowing the measurement of phosphorescence at room temperature. One
approach is to place a drop of solution containing the analyte on a small filter paper
disk mounted on a sample probe. After drying the sample under a heat lamp, the
sample probe is placed in the spectrofluorometer for analysis. Other solid surfaces
that have been used include silica gel, alumina, sodium acetate, and sucrose. This
approach is particularly useful for the analysis of thin-layer chromatography plates.

10G.3 Quantitative Applications Using Molecular Luminescence

Molecular fluorescence and, to a lesser extent, phosphorescence have been used for
the direct or indirect quantitative analysis of analytes in a variety of matrices. A di-
rect quantitative analysis is feasible when the analyte’s quantum yield for fluores-
cence or phosphorescence is favorable. When the analyte is not fluorescent or phos-
phorescent or when the quantum yield for fluorescence or phosphorescence is
unfavorable, an indirect analysis may be feasible. One approach to an indirect
analysis is to react the analyte with a reagent, forming a product with fluorescent
properties. Another approach is to measure a decrease in fluorescence when the an-
alyte is added to a solution containing a fluorescent molecule. A decrease in fluores-
cence is observed when the reaction between the analyte and the fluorescent species
enhances radiationless deactivation, or produces a nonfluorescent product. The ap-
plication of fluorescence and phosphorescence to inorganic and organic analytes is
considered in this section.

Inorganic Analytes Except for a few metal ions, most notably UO2+, most inor-
ganic ions are not sufficiently fluorescent to allow a direct analysis. Many metal OH HO
ions can be determined indirectly by reacting with an organic ligand to form a
HO N N
fluorescent, or less commonly, a phosphorescent metal–ligand complex. One ex-
ample of a chelating ligand is the sodium salt of 2,4,3′-trihydroxyazobenzene-5′-
alizarin garnet R SO3Na
sulfonic acid, also known as alizarin garnet R, which forms a fluorescent
metal–ligand complex with Al3+ (Figure 10.47). The analysis is carried out using (a)
an excitation wavelength of 470 nm, with fluorescence monitored at 500 nm.
O
Other examples of chelating reagents that form fluorescent metal–ligand com- Al3+
plexes with metal ions are listed in Table 10.12. A few inorganic nonmetals are O
N
determined by their ability to decrease, or quench, the fluorescence of another
HO N
species. One example is the analysis for F–, which is based on its ability to quench SO3Na
the fluorescence of the Al3+–alizarin garnet R complex.
fluorescent complex
Organic Analytes As noted earlier, organic compounds containing aromatic (b)
rings generally are fluorescent, but aromatic heterocycles are often phosphores- Figure 10.47
cent. Many important biochemical, pharmaceutical, and environmental com- Structure of (a) alizarin garnet R, and (b) its
pounds are aromatic and, therefore, can be analyzed quantitatively by fluorometry metal–ligand complex with Al3+.
1400-CH10 9/8/99 4:19 PM Page 430

430 Modern Analytical Chemistry

Table 10.12 Selected Chelating Agents for the Fluorometric

Analysis of Inorganic Metal Ions
Chelating Agent Metal Ions

8-hydroxyquinoline Al3+, Be2+, Zn2+, Li+, Mg2+ (and others)

flavonal Zr2+, Sn4+
benzoin B4O72–, Zn2+
2′,3,4′,5,7-pentahydroxyflavone Be2+
2-(o-hydroxyphenyl) benzoxazole Cd2+

Table 10.13 Selected Examples of Organic Compounds of

Biochemical, Pharmaceutical, and Environmental
Significance That Show Natural Fluorescence or
Phosphorescence
Class Compoundsa

aromatic amino acids phenylalanine (F)

tyrosine (F)
tryptophan (F, P)
vitamins vitamin A (F)
vitamin B2 (F)
vitamin B6 (F)
vitamin B12 (F)
vitamin E (F)
folic acid (F)
catecholamines dopamine (F)
norepinephrine (F)
pharmaceuticals and drugs quinine (F)
salicylic acid (F, P)
morphine (F)
barbiturates (F)
LSD (F)
codeine (P)
caffeine (P)
sulfanilamide (P)
environmental pollutants polycyclic aromatic hydrocarbons:
pyrene (F)
benzo[a]pyrene (F)
organothiophosphorous pesticides (F)
carbamate insecticides (F)
DDT (P)

aAbbreviations:F = fluorescence, P = phosphorescence, LSD = lysergic acid diethylamide,

DDT = dichlorodiphenyltrichloroethane.

or phosphorometry. Several examples are listed in Table 10.13. When an organic ana-
lyte is not naturally fluorescent or phosphorescent, it may be possible to incorporate it
into a chemical reaction that produces a fluorescent or phosphorescent product. For
example, the enzyme creatine phosphokinase can be determined by using it to catalyze
the formation of creatine from phosphocreatine. The creatine that is formed reacts
with ninhydrin, producing a fluorescent product of unknown structure.
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Chapter 10 Spectroscopic Methods of Analysis 431

Standardizing the Method Equations 10.32 and 10.33 show that the intensity of
fluorescent or phosphorescent emission is proportional to the concentration of the
photoluminescent species, provided that the absorbance of radiation from the exci-
tation source (A = εbC) is less than approximately 0.01. Quantitative methods are
usually standardized using a set of external standards. Calibration curves are linear
over as much as four to six orders of magnitude for fluorescence and two to four
orders of magnitude for phosphorescence. Calibration curves become nonlinear for
high concentrations of the photoluminescent species at which the intensity of emis-
sion is given by equation 10.31. Nonlinearity also may be observed at low concen-
trations due to the presence of fluorescent or phosphorescent contaminants. As dis-
cussed earlier, the quantum efficiency for emission is sensitive to temperature and
sample matrix, both of which must be controlled if external standards are to be
used. In addition, emission intensity depends on the molar absorptivity of the pho-
toluminescent species, which is sensitive to the sample matrix.
Representative Methods

Method 10.3 Determination of Quinine in Urine23

Description of Method. Quinine is an alkaloid used in treating malaria (it also is
found in tonic water). It is a strongly fluorescent compound in dilute solutions of
H2SO4 (Φf = 0.55). The excitation spectrum of quinine shows two absorption bands
at 250 nm and 350 nm, and the emission spectrum shows a single emission band at
450 nm. Quinine is rapidly excreted from the body in urine and is easily determined
by fluorescence following its extraction from the urine sample.

Procedure. Transfer a 2.00-mL sample of urine to a 15-mL test tube, and adjust
the pH to between 9 and 10 using 3.7 M NaOH. Add 4 mL of a 3:1 (v/v) mixture of
chloroform and isopropanol, and shake the contents of the test tube for 1 min.
Allow the organic and aqueous (urine) layers to separate, and transfer the organic
phase to a clean test tube. Add 2.00 mL of 0.05 M H2SO4 to the organic phase, and
shake the contents for 1 min. Allow the organic and aqueous layers to separate, and
transfer the aqueous phase to the sample cell. Measure the fluorescent emission at
450 nm, using an excitation wavelength of 350 nm. Determine the concentration of
quinine in the urine sample using a calibration curve prepared with a set of external
standards in 0.05 M H2SO4 and a distilled water blank. The external standards are
prepared from a 100.0-ppm solution of quinine in 0.05 M H2SO4.

Questions
1. Chloride ion is known to quench the intensity of quinine’s fluorescent
emission. For example, the presence of 100 ppm NaCl (61 ppm Cl–) gives an
emission intensity that is only 83% of that without chloride, whereas the
presence of 1000 ppm NaCl (610 ppm Cl–) gives a fluorescent emission that is
only 29% as intense. The concentration of chloride in urine typically ranges
from 4600 to 6700 ppm Cl–. How is an interference from chloride avoided in
this procedure?

Extracting the aqueous urine sample with a mixture of chloroform and

isopropanol separates the quinine and chloride, with the chloride remaining in
the urine sample.

2. Samples of urine that do not contain quinine still contain a small amount of
fluorescent material after the extraction steps. How can the quantitative
procedure described earlier be modified to take this into account?

—Continued
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432 Modern Analytical Chemistry

Continued from page 431

One approach is to prepare a sample blank using urine known to be free of

quinine. The fluorescent signal for the sample blank is subtracted from the
urine sample’s measured fluorescence.

3. The fluorescent emission for quinine at 450 nm can be induced using an

excitation frequency of either 250 nm or 350 nm. The fluorescent quantum
efficiency is known to be the same for either excitation wavelength, and the
UV absorption spectrum shows that ε250 is greater than ε350. Nevertheless,
fluorescent emission intensity is greater when using 350 nm as the excitation
wavelength. Speculate on why this is the case.

From equation 10.32, If is a function of k, Φf, P0, ε, b, and C. Since Φf, b, and C
are the same for either excitation wavelength, and ε is larger for a wavelength
of 250 nm, the greater If for an excitation wavelength of 350 nm must be due to
larger values for P0 or k at a wavelength of 350 nm. In fact, P0 at 350 nm for a
high-pressure Xe arc lamp is about 170% of that at 250 nm. In addition, the
sensitivity of a typical photomultiplier transducer (which contributes to the
value of k) at 350 nm is about 140% of that at 250 nm.

10G.4 Evaluation
Scale of Operation Molecular photoluminescence can be used for the routine
analysis of trace and ultratrace analytes in macro and meso samples. Detection limits
for fluorescence spectroscopy are strongly influenced by the analyte’s quantum yield.
For analytes with Φf > 0.5, detection limits in the picomolar range are possible when
using a high-quality spectrofluorometer. As an example, the detection limit for qui-
nine sulfate, for which Φf is 0.55, is generally between 1 ppb and 1 ppTr (part per
trillion). Detection limits for phosphorescence are somewhat poorer than those for
fluorescence, with typical values in the nanomolar range for low-temperature phos-
phorometry and in the micromolar range for room-temperature phosphorometry
using a solid substrate.

Accuracy The accuracy of a fluorescence method is generally 1–5% when spectral

and chemical interferences are insignificant. Accuracy is limited by the same types
of problems affecting other spectroscopic methods. In addition, accuracy is affected
by interferences influencing the fluorescent quantum yield. The accuracy of phos-
phorescence is somewhat greater than that for fluorescence.

Precision When the analyte’s concentration is well above the detection limit, the
relative standard deviation for fluorescence is usually 0.5–2%. The limiting instru-
mental factor affecting precision is the stability of the excitation source. The preci-
sion for phosphorescence is often limited by reproducibility in preparing samples
for analysis, with relative standard deviations of 5–10% being common.

Sensitivity From equations 10.32 and 10.33 we can see that the sensitivity of a flu-
orescent or phosphorescent method is influenced by a number of parameters. The
importance of quantum yield and the effect of temperature and solution composi-
tion on Φf and Φp already have been considered. Besides quantum yield, the sensi-
tivity of an analysis can be improved by using an excitation source that has a greater
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Chapter 10 Spectroscopic Methods of Analysis 433

From
source

(a) To
detector

From
source

Figure 10.48
Use of slit orientation to change the volume
To from which fluorescence is measured:
(b)
detector (a) normal; (b) rotated 90°.

emission intensity (P0) at the desired wavelength and by selecting an excitation

wavelength that corresponds to an absorption maximum (ε). Another approach
that can be used to increase sensitivity is to increase the volume in the sample from
which emission is monitored. Figure 10.48 shows how a 90° rotation of the slits
used to focus the excitation source on the sample and to collect emission from the
sample can produce a 5–30-fold increase in the signal.

Selectivity The selectivity of molecular fluorescence and phosphorescence is

superior to that of absorption spectrophotometry for two reasons: first, not
every compound that absorbs radiation is fluorescent or phosphorescent, and,
second, selectivity between an analyte and an interferant is possible if there is a
difference in either their excitation or emission spectra. In molecular lumines-
cence the total emission intensity is a linear sum of that from each fluorescent
or phosphorescent species. The analysis of a sample containing n components,
therefore, can be accomplished by measuring the total emission intensity at n
wavelengths.

Time, Cost, and Equipment As with other optical spectroscopic methods, fluores-
cent and phosphorescent methods provide a rapid means of analysis and are capa-
ble of automation. Fluorometers are relatively inexpensive, ranging from several