Nomenclature, symbols, units and their usage in spectrochemical analysis—VI molecular luminescence spectroscopy

.N

SPECTROSCOPY

CONTENTS I.

1NTR~~~~~~~~~

2.

~~~IN~T~ONOF L~~NESCE~C~ AND ~A~T~~S

USED XN PARESIS

2.1 Types of luminescence 2.2 Absorptionand deactivation processes Absorption 2.2,1 transitions 2.2.2 Radiationless 2.2.3 Radiativetransitions 2.2-4 Hatrixeffects Table 2.1 and Figure2,l Schematicdiagramof radiative,radiationless vibrationalrelaxationbetweenelectronic statesin a r-electronicsystem 3,

rNSTR~~~TA~ paces 3.1 3,2 3.3 3.4 3.5

Excitationsources Opticalsystems Detectors Modulationof the opticalsignal Polarizers

Table 3.1 Terms, symbolsand units for the excitation and detectionof the analytical signal Figure3.1 Examplesof typesof l~inesce~ce S~ctrometeTs 4,

~ASU~~~T 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8

ANI USE OF L~~~~SCE~~E Phone

IN ANALYSlS

parameters Classification of luminescence Emissionspectra Excitationspectra Excitation-emission spectra Lifetimesof luminescence Quantumyields Polarization of luminescence Quantitativeanalysis

to radiant Table 4.1 Terms,symbolsand units relating energyand its interactionwith matter Table 4.2 Classification and symbofsfor luminescence parameters 5,

FACTORSAFFECTER&LUMl~ES~~N~~ DATA 5.1 5.2 5.3 5.4

Geometricarrangementof the sample k-e-filter,post-filterand self-absorption effects Refractioneffects Solventand temperatureeffects

260

261

INTRODKTXON

1.

Part

Vl

IWhC

_

their

is

a sequel

to previous

documents in the series “Nomenclature, Symbols, Units and issued by the Analytical Chemistry Division of Analysis”

Usage in Spectrochemical

This document does not aim to be completely self-contained since many of the terms and units needed for describing Molecular Luminescence Spectroscopy have already appeared in Parts I, all terms important to Molecular Luminescence, However to facilitate reference, II and III. together with their symbols and units - and these include many appearing in previous documents - are presented in the Tables. In the past, the terms quantum yield and quantum efficiency have usually been considered in terchangeab 1e _ It is now recommended that these terms should be used strictly as defined in Section 4.6. In part Vi, the use of photon quantities is presented for the first time in these series. Photon quantities are important in Molecular Luminescence Spectroscopy and although they have organization has come forward with recommenbeen in use for some years, no international Where the measurement is primarily interested in dations for symbols for these quantities. it is recommended that a subscript p the number of photons flowing in a beam of radiation, be used on the corresponding energy or flux quantity (see Section 4.2 and Table 4.1). 2.

DEFINITIONOF LUMINESCENCE AND PABAMETERS USED IN ANALYSIS

2.1 Types of luminescence The various types of molecular luminescence observed can be classified by (a) the mode of excitation tog the excited state capable of emission and (b), the type of- molecular excited state (Table 2.1). Fluorescence is the spin-allowed radiative transition while pbosphores-

cence is the result of a spin-forbidden radiativetransition. TABLE2.1

Classification

(a) excitation

of radiation

(UV/VIS)

type

photoluminescence chemiluminescence, bioluminescence

reaction

thermally activated recombination injection

luminescence

mode

absorption chemical

of types of luminescence

thermoluminescence

ion

electroluminescence

of charge

high energy particles radiation

radioluminescence

or

friction

triboluminescence

sound waves

sonoluminescence

(b) excited state (assuming ground singlet state) first lowest

excited

singlet

triplet

luminescence

type

fluorescence, delayed fluorescence

state

phosphorescence

state

Fluorescence, delayed fluorescence and Phosphorescence can also arise from excited states higher than the first and therefore the transition should be indicated bv a subscrint, However the quantum yield of radiative processes from higher excited stales are gen;?rally several orders of magnitude lower than the quantum yields of emission from the first excited state. Therefore if no special indication is given, the quantum yields are those of the respective first excited states. Three types of delayed

fluorescence

are known:

(i) E-type deIayed fluorescence: The first excited singlet state becomes populated by a thermally activated radiationless transition from the first excited triplet state. Since in this case the population of the singlet and triplet states are in thermal

262

CO~lXISSION ON SPECTROCHEMICAL

the

equilibrium, are equal.

Ii fetimes

of

delayed

fluorescence

ANAl,YSIS

and the concomi tnnt

phosphorcsccnce

(ii) P-type delayed fluorescence: Ihe first excited singlet state is populated by interaction of two molecules in the triplet state [triplet-triplet annihifation) thus producing one molecule in the excited singlet state. In this biphotonic process the lifetime of delayed fluorescence is half the value of the concomitant phosphorescence. (iii) Recombination by recombination of

opposite

fluorescence: radical ions

The first excited singlet state electrons or by recombination

with

charge.

becomes populated of radical ions of

Whereas delayed fluorescence rarely has analytical applications, fluorescence and phosphorescence are of practical importance in luminescence analysis. Absorption of light is the preferred mode of excitation while chemiluminescence (production of luminescence radiation by chemical reaction) plays a minor role. The other excitation modes listed in Table 2.1 do not prove to be useful in analysis. 2.2 Absor tion and deactivation recesses In principleradiativeandradiationlesstransitions can be distinguished in molecules. Ihe first occurs by absorption -goflight quanta, and the latter is the result of the transformation of electronic excitation energy into vibrational/rotational energy. In both radiative and radiationless transitions the principle applies that transitions between terms of the same multiplicity are spin-allowed while transitions between terms of different multiplicity are spin-forbidden (spin conservation rule). The intercombination prohibition for transitions between terms of different multiplicity in molecules becomes more relaxed the more efficiently spin-orbit coupling (jj coupling) perturbs the wavefunctions of the pure states into wavefunctions of mixed spin states. As a result, spin-forbidden transitions can sometimes compete with spin-allowed transitions. Generally, the transition probabilities of radiationless transitions are higher, the smaller the energy difference between the ground vibrational levels of the electronic states that are involved in the transition. The definitions of the various radiative and radiationless cules are illustrated in the term scheme in Fig. 2.1.

transitions

which occur

in mole-

Singlet-singlet absorption results in the transition from the 2.2.1 Absorption. singlet ground state of the molecule into singlet excited states (So + Sn) and leads to the UV/VIS absorption spectrum. The analogous triplet state triplet-triplet

triplet-triplet absorption takes place with the transition from the lowest of the molecule to higher triplet states (‘I1 + Tn) thus leading to the absorption spectrum.

Singlet-triplet absorption takes place with the transition from the singlet the molecule to triplet states (So + T,,) and results in the singlet-triplet spectrum.

ground state absorption

of

Each absorption transition is characterized by the energy of the absorbed radiation, the oscillator strength and the polarization of the transition as well as the vibrational The oscillator strength depends on the multiplicities of the structure of the band system. their orbital character (n, ?I* or n, n* states) and on the participating electronic states, symmetries of the initial and final states. knowledge of the IJV/VIS absorption spectra of the compounds studied is of particular In this context it has to be taken into account that importance in luminescence analysis. fJV/VIS absorption spectra measured in a solid matrix at low temperatures are generally different from spectra measured in fluid solution at room temperature. Smaller half-widths of the bands and higher molar absorption coefficients of the absorption maxima are invariably observed in the solid matrix. The

Intrachro~phor~c radiationless transitions take 2.2.2 Radiationless transitions. interchromophoric radiationless transitions pIace within the term system of the molecule, between the radiationless

term systems transitions

Interchromophoric transfer

processes.

of two non-conjugated between two molecules

and intermolecular

radiationless

parts of the of identical transitions

molecule, intermolecular or different species. are

electronic

energy

PROViSTON&:

NawncZatutt?

in molecular

luminescence

263

S&WXtWSCQ&?y

Intracbro~~bori~ radiationless transitions between Sta?xs of the same rn~~~~~~ic~t~ are named internal conversion (IC}: Sn--+Sl, Sl --*So, Tn --+Tl being distinguished. I~trac~ro~~b~r~c radiationless transitions between states of different multiplicity named intersystem - crossing (IX): Sl --*Tn, Tl --*So, Tl --+%.I are known.

are

(spin-aX towed), electronic energy transfer processes are known: singlet-singlet singlet-tripfet (s~io~farb~dde~~ and triplet-singlet triplet-triptet (spin-allowed), transfer (shim-forbid~en~.

Ihe fo~louing

The most important property oE radiationless transitions for analytical work is the transition probability because this determines the yield of luminescence. The quantum yields of fluorescence YF and phosphorescence Yp are related to the radiative and radiationless rate constants as follows: YF =

uhere the rate constants

rare ixmstaltt

relate

kFH kFH + km + kGm

to the transitiatts

as follows:

transition

ISC (S, -4,) ISC (TI --+ So, TC

(S, --“SJ

~hos~bores~en~e LusGnescence quenching is defined as the radiationless redistribution af the exeitatian energy via interaction (electronic energy or charge transfer) between the emitting species and the quencher. Quencher and emitter can be molecules of the same species (~oncent~tio~ quenching] 5t of different species. ‘Ihe deactivation of the priloarily excited emitter can Iead to tha activation of the cpmcher followed by radiative deactivation (sensitized luminescence) * In some cases concentration quenching is accompanied by the formation of a uew bimolecular species which is capable of emission (excimer- and exc~~~ex-~~iRescence~* In special selectivity

cases ~~i~es~en~e quenching effects can be used to enhance sensitivity in the luminescence analysis of mixtures:

(i) The abserved rate constants perturbers axe often significantly compounds, for example isomers.

kq of fIuorescence q~~~~bi~g by external even in the case of closely different

(ii) The strong depopulation of the fluorescing atom perturbers can lead to a large population state [enhanced phosphorinetry ).

and/or

heavy atom

related

singlet excited state by external heavy of the phosphorescing triplet excited

(iii) In general, strong eLectron accept5rs quench the fluorescence of alternant pakycyclic aromatic hydrocarbons more efficiently than the fluorescence of the nonalternant systems and the reverse effect takes place with strong electron donors as fluorescence quenchers. The application of the effects mentioned in (i’l and (iii) in luminescence analysis are examples af the technique of quenched fluorimetry. The use of the terms ‘~en~an~opbo5~~~~~metry” and ?~quen~hof~~ri~try’~ is not recommended. 2.2.3 Radiative transitions. As to the definition cence and phosphorescence see Section 2.2 and Fig. 2.1.

of fluorescence,

delayed

fluores-

Fluorescence radiation occurring at wavelengths Ionger than absorption, E.e., the normal case t is said to be of the Stokes type. Fluorescence radiation occurring at shorter wavalengths than absorption is classified as the anti-Stokes ty&e.

IC

S*

0, ,

P

I

A/R t

t 8 f

%I_; FIG. 2.1

Schematic diagram of radiative (solid vertical lines), radiationless (wavy horizontal lines), and vibrational relaxation (broken vertical lines) between electronic states in a a-electronic system.

States:

So

= ground state, Sl = first excited singlet state, Sg = second excited singlet state, T1 = lowest triplet state, and Tn = excited triplet states.

Transitions: A = absorption (S,+Sn, S,+Tl, Tl+T,,),XC = internal conversion (Sn--41, Sl--+So, Tn--+Tl). ISC = intersystem crossing (Sl---CT,,T1--6,). VR = vibrational relaxation, F = fluorescence (Sl+So), and P = phosphorescence (Tl+So)

FIG. 3.1

Examples of types of luminescence spectrometers. (a) single-beam, (b) double-beam, (c) double(spectral) beam, (d) double-(s~ci~r~nous)beam. X = excitation bcnm, S = sample cell, OS = beam splitter, BA = beam alternator, fibI = emission monochrormrtor,EX = excitation monochromator, f'= phoroJctector, and FI= w~lvclcngthdrive

Nomenclature

PROVISIONAL:

The following characteristic luminescence analysis:

parameters

(if

the luminescence

spectrum

(ii)

the luminescence

quantum yield

(iii)

the luminescence

lifetime

(see Sections

4.1.

of

radiative

transitions

arc

the

most

important

in

4.3 and 4.4).

Phosphorescence quantum yields sufficient for analytical a~~l~licat~ons are generally obtained only if bimolecular radiationless deactivation of the phosphorescing triplet state is avoided by carrying out the measurements in a solid matrix (at low or rOOmtemperature) or by carrying out the measurements with the substance in the adsorbed state (at low or room Room temperature phosphorescence in liquid solution can be applied to temperature). analysis provided the solution is efficiently deoxygenated. If the luminescence lifetimes of the different species of a mixture to be analyzed differ time-resolved luminescent ~a$ure~nts can be used for analytical purposes. sufficiently, 2.2.4 analysis:

The following

Matrix effects.

matrix effects

are important in luminescence

- Addition of acid or base to the solution of a fluorescing (i) Acid/base interaction or phosphorescing compound which contains functional groups with dissociatabie protons or lone or non-bonded electron pairs can lead to spectral shifts by protonation. In some cases aromatic molecules having non-bonded electron pairs fail to fluoresce in non-activating solvents because the lowest excited singlet state is of the n, 1~* type ‘lhe addition of small amounts of acid which usually favours intersystem crossing. results in proton&ion with the non-bonded pairs often raising the energy of the n, n* to such a degree that the lowest x, x* state becomes the lowest excited singlet state, making fluorescence likely. (ii) - In so-called Shpol’skii matrices, especially alkanes, in whit the dissolved and the solvent molecule are similar, fluorescence (phosphorescence) spectra at low temperature are often characterized by a very large number of bands with very small half-widths. Such spectra are useful fox the identification of compounds. (iii) External heavy atom effects - If compounds with elements which have a large Z-number (heavy atoms) are present in the matrix there can be generally observed a decrease of fluorescence quantum yield and fluorescence lifetime, an increase of phosphorescence quantum yield, a decrease of phosphorescence lifetime and in some cases characteristic changes of the vibrational structure and relative intensity distribution of the phosphorescence spectrum. These external heavy atom spin-orbit coupling effects are useful to enhance sensitivity and/or selectivity in luminescence analysis (see Section 2.2.2).

.

Para~~etic cempounds - Paramagnetic substances which are present in the matrix enhance spin-orbit coupling in the luminescinn compound. Therefore in general they cause luminescence effects-of the same kind as-observed with heavy atom perturbers _ [iv)

(see above). INSTR~NTA~ PARAMETERS

3,

‘Ihe instrument used to measure luminescence emission spectra is termed a luminescence (fluorescence, phosphorescence) spectrometer. (See Nomenclature, Symbols, Units and their Usage in Speetrochemical Analysis, Part III). 3.1 Excitation source Generally in luminescence spectroscopy a high flux of radiation (the excitation source) is needed for the excitation of the analyte and metal vapour or gas discharge lamps are commonly used. For a discussion of various radiation sources, see Part V of this series. Flash lamps, i.e.,lampswhich which

give

a short

contain pulse, are

output

an inert gas which can be rapidly pulsed, useful for determining short luminescence

or lasers deca-s,

3.2 Optical systems ‘Ihe selection of radiation of the required wavelength from the excitation source for exciting the analyte may be achieved with filters or with an excitation ~nochromatar entrance

and exit

slits

to

givr

the

required

spectral

band width

(see

Part

X, Section

using 5).

Luminescence radiation of the required ~a~e~e~~~~~is selected from the sample by an emission is used for excitation and a single beam of man~chro~~~or. Where :I single beam of radiation l~~incscenec radiation is t&en from the sample, the instrument would ba termed a singlebeam ~iu~~~nes~ci~~e~spcct~romctor. ruble-beam spectrometers are used for irn?~ro~~n~ stability and for the direct mcnsuremont of excitation spectra. Double (spcctralt beam spectrometers arc used where two samples arc to be excited by two different wavelengths. R double ~s~chr~~~~~as~ beam spectrometer is a luminescence spectrameter in which both the excitation and emission m~no~hromators scan the excitation and emission spectra simultaneously, usually with 3 fixed wavelength difference between excitation and cmisskon. Examples of the four types of Iuminescence spectrometers are shown on Fig. 3.X. 3.3 Photodetectors Ihe pb~~~~~tip~~er tube using single photon c0~n~in~ or current m~asure~n~, is the mrst satisfactory detector far measuring luminescence emission, Other detectors are often used in luminescence spectroscopy far monitoring the energy or photons in the excitation beam and for calibrating procedures. Ther~~piles (series connected ~hermoco~~~es attached to a blackened callector surface), bolometers [thin b~a~k~R~d collector with a hi ~emp~~~~~@ coefficient of resistance) and pyroelectric detectors {based on the temperature dependence of ferroe~~~riG~ty in some crystals) are detectOr wbicb produce an electrieaf signal proportional to the energy flux on the collector surface. @anturn counters produce an efectrical signal ~ro~or~ion~~ to the photon flux absorbed in a flwtrescent solution. Chemical actinometers axe detectors in which the amount of a chemical product Earmed is proportional to the numbers of photons absorbed. Silicon photodiodes may be used either in the p~~~ovol~a~~ or PhotocOndu~tive mades for measuring radiation fXuxes and, although less sensitive &an ~bo~ornu~~i~~~~rs, their gain stability is very good, Image devices fvidicons,pbotodiode arrays,etc.) metry especially for fast acquisition Of data,

am sonretimes used in ~~~~OScen~~ spectra-

Where pbotodeteetors are Switched On for off) usually in a repetitive manner e~~o~i~g electronic switches, they are termed gated ~bO~Ode~e~~OrS_ 3.4 chanical Or electronic means to give an inWSity The equency ~d~la~~o~ is used in addition, for eaSe in modulated beam. Mten amPXitude OT signal processing, i&ted pbo~~e~a~tors /Section 3.3) are ~re~~en~l~ used in ~on~~~~~on with inodulatedlight to baron the Signallnoise ratio, %O separate f~~ox~SGO~~e from pbospbOreScence or ta measureluminescencedecay times. are mechanical devices used to separate phosphorescencefrom fluorescence. derivative id~~~d~? of the Eminescence spectrum is radiation may be achieved by, for exaaple, rotating 3.5 Polarizers A plane polarizer 3;san OptIcal device which alXows the transmission of IWiiatiOn Of which the electric vector is restricted to one plane resulting in plane polarized radiation. TABLE

5.1.

Tmrs,

s

detection

01s and units for thtllg excitation of the analytical signal

Terms

SymboXs

entrance fexitl 51i2width of ~onocbro~to~ entrance (exit) sfitheight of ~~ochro~to~ spectral

ba~dwi~h of mom-

chromator(if the excita~iW% ~noc~romatox is of Concern, replace m with ex and if the emission ~*o~br~~t~r iS of concern, replace with em) 10% (or 3%) ba~dwid~b Of spectral filter

spectral radiant flux of source at wavelength h

and

Practical units

5

mu

h

mm nm

NoteS See Pax% I

tl

lk

+t

~aye~e~~~h my be replaced by wavenumber or frequency

See

Part “I11

267

Terms

SymboIs

Cr;,Rsnrittance of excitation

Tcx(M

monochrornator to non-polarized radiatiotl at wavelength X (if the emission monochromntor is ex by em) of concern, replace conductance

optical

photodetector at wavelength

response h

solid angle over which radiation is absorbed in the cell solid

PractiWl

units

1

See

2 m sr

G YOl

A w-1

aA

51:

I II: and

See Part 1, Section 5.3.2

F denotes

fluorescence, P phosphorescence, DF delayed fluorescence

angle over which is measured

phase of ac modulated fluerescence or phosphorescence or delayed fIuorescence with respect to the modulated exciting radiation

rart

TabIc 4.1

luminescence

degree of modulation (m = ratio of ac component to dc component)

NOtlCS

For the exciting radiation use subscript ex

=F(P,DF) e

degrees

delay time between term& nation of exciting radiation and measurement of fIuorescence (phosphorescence, delayed fluorescence) excitation “on-time”

time [source per cycle)

observation time (detector oon-time*7 per cycle) cycIe time (sum of the time for excitation and observation including delay times = tF +t Il + t0 + t*$ 4.

~S~~~

Ah% USF QF L~I~ESCE~~

P~~E~~S

IN ~~LYSIS

4.I Classification of luminescence parameters ?%e lumanescence property of an analyte as measured by the appropriate instrument will often be distorted by instrumental and sample effects and the property would be referred to as the measured luminescence parameter. Corrected parameters are those derived by correcting the measured parameters for instrumental artefacts, for post-filter effects and other sample effects (see Section 5). Table 4.1 lists the luminescence parameters and the symbols used. TABLE4.1

Terms, symbols and units relating to radiant energy and its interaction with matter

Terms (radiant]

Symbals

Practical

Q

J

energy

QP spectral energy

(radiant)

QX

= dQfdk

units

Notes See Part I

photons J nm-1

See Fart III (photon quantity)

263

COMMLSSION ON SPECTROCHENTCAL ANALYSIS ‘Terms

Symbols

radiance

H,

(radiant) density radiant

energy

radiant intensity at time t after termination of excitation (radiant)

energy flux

I.

J m-3

I

W sr

I(O)

w sr

I(t)

w sr-1

at

spectral. (radiant) energy flux

P,

/dt

4j = d#/dX

-1 -I

number of_l photons s

PlX

(photon quantity)

-1 N nm

= dQlp/dh number of_l photons s

Q

rotcs

w

@ = dQ/dt @p = d

units

-2 -1 Wm sr

u, w

intensity

radiant intensity time t = 0

Practical

-1 nm

(photon quantity)

radiant flux incident on (absorbing) medium radiant flux transmitted by (absorbing) medium radiant flux reflected by sample radiant flux absorbed by medium transmittance reflectance absorptance absorptivity

or

internal

transmittance

internal

absorptance

internal

absorbance

Transmittance of medium itself disregarding boundary effects

A =

molar absorption coefficient

integrated absorption

molar coefficient

absorption

path length

molar concentr:ttion of absorber

1

-log ri

1

K

-1 em

1

(linear) absorption coefficient

A value at the wavelength peak (Xo)

a.

mol

E

AGo?

C

mall’

cm-l

cm -1

tn

Also called molar lineic absorbance

1

dX

1, b

-1 cm

-1

mol II

An additional subscript can be used to denote the species

269

waveiengrh at band pcnk

uavelerrgth of fkorcscence fpbusphorescance, delayed fluorescence) quantum yield of Eluorescence ~~bospb~~es~eoce, delayed fluorescence)

x

0

h F(P,lG)

YF[r,nF)

nm

nm

x can be replaced by ii(~avonumber) or v ffreqrWlcyl

1

PM? symbof Y conforms with Part IXX and is recommended over previously trsed symbof s

energy yield of fluorescence (~b~s~horesce~~e, delayed fluorescence) quota efdciency of fltuorescence(phosphorescence) lifetimeof fluorescence (phasphorescence, delayed fluorescence)

See

sccrion 4.5

dissociation~o~s~an~(acidbase) of moleculein first excitedsingletstate (= CH+CA-*/C~* in equilibrium at temperatureT) dissociationconst*t (acidbase) of smlesulle in &West tripletstate (= c~~c~_*~~~~ in equiiibrium at temperature T) radiant intensitiesof the beam resolvedinto directions paralleland ~r~nd~~u~ar to the dixection af polarization of the excitingradiation degree of polarization degxee of polarization (correctedfor depolarizing factors)

P = [I

p*

degree of de~~ar~za~~o~or dichroicemissionratio degree of anisotropy 42

Emissionspectra The measuredemissionspectrumof a sample is the spectrumas obtainedfrom the instr~nt. The correctedemissionspectrumis obtainedafter correcting for instrumental and sample effects and 1s ~suaZly representedby a graph of (nh(see Table 4.1) againstwavelength. @x may be transformed to other quantities as follows: wavelength

scale

(nn) ;

wht?rc

N

4.3 Exciratiort spectra The spectrum observedby ~~~as~r~n~ the variationof the ~~m~~es~ence flux from an analyrrBS

a functionof the excitationwavelengthis termeda measured(fluorescence, ~~~s~~orcs~ence~ excitationspectrum. A corrected excitation spectrumis obtaiilcd if the photon flux incident Kthc sample is held constant. If the solution is suFficicntfv diIutc that the fraction of the excitingradiationabsorbedis propdrtional to the absorptioncoefficientof the analyte,and if the quantumyield is ~r~de~endent of the excitingwavelength,the correctedexcitationspectrumwill be identicalin shape to the absorptionspectrum. 4.4 Excitat%on-emission spectra +%e three-dimensional spectrumgeneratedby scanningthe emissionspectrumat ~ncre~ntai steps of excitationwavelerrgth (x axis = emissionwavelength,y axis = excitation wavelength. z axis = emissionflux) is called a (fluorescence, ~~o~phorescen~e~ excitation-emission 53~CtTUm(or EESj (NoFe I). These spectraare ~ar~ic~~ar~yuseful for investigating samples~o~t~in~ngmore than one emittingspecies. CorrectedEES are obtainedif (a] the emissionis correctedfo~linstru?nental response With wavelength,and (61 the excitjngradiationflux in photonss is held constantfor all excitationwavelengths. A e correspondsto the curve where a plane,parallelto the z-axis,

obtained by varyingboth the sionalspectrumwhich intersectsthe EES.

The lihtime of iumimscence is define?as the time requiredfor the luminescence intensity to decay from some inittalvalue to e- af that value (e = 2.718...). Lifetimescan be measuredby @ase f~~or~~try (pbo~~hori~t~) where the phase shift betweenthe sinusoidalfy xlodulated exertinglightand the emittedfight is measured. Flash fluorimetry(phosphorimetry] is the term used when decay times of ~~~nes~e~ce are ~as~red using a pulsed source 2j?F"ZZiation. It is often necessaryto separatethe signafdue to the light flash from lumktescenceemissionsignalby a deconvo~ution technique in order to obtain the correct decay curve for emission. Decay times correctedEOT this erfect are termedcorrecteddecay timer of fluorescence or ~ho~~~o~~~ence.

4.6 @anturnyields 'Theq~nt~rnyield of ~~~ne~~en~e of a speciesis the ratio of the Noel of photos emttted to the number of photonsabsorbedby the sample. Ibe measuredquantumyield of luminescence (fluorescence or ~~~spbores~e~~e~ is the measurementmade with a fluorescence(phosphorescencefspectrometer when no correctionsare made for instrumental responseor for sample effects. Ihe correctedquantumyield of luminescence is obtainedwhen the measuredquantum yield is correctedfor instrumental response,pre- and post-filtereffectsand refractive index effects. The energyyield of luminescence of a speciesis definedas the ratioof the energyemitted as luminescence to the energyabsorbed by the species. ~ntum

yields

aE

q~nching by other the Stem-Volmer

~~~~~~s~e~~e

species law:

y. -y-

(~~uspboresc~~ce~ of an ~~alyt~are often reduceddue to wenching processes generally follow in the analytic solution.

1 = kQ CQ ~~

luminescence

yield

in the absence bf quencher Q

luminescence

yield

with quencher of concentration

rate constant luminescence

c

for quenching lifetime

in the ahserrcc of quencher

Q

Q

Polarizntion nf emission is not of great: importance in molecular iumincsccnce q’ectroscopy Measurement of potaritatian is nurlcliiy made at unless the solvent used is viscous or solid. rip&t

:~ngXes

to

the

direction

of

propagation

of

the

exciting

radiation

and must

take

account

lhe relations noIarir.:%tion effects of all oncical camnonents in the instrument. R, and the degree uf hctwcetr^ the degree of polarization P: the degree of depolarization anisotropy r, (for definitions set Table 4.1) are: of

the

corrected l~im~ncsceuce excitation ~o~a~~zatio~ spec~~m of an analyte is o~?~a~~ed when Since this specthe polarization is measured as a function of the excitation wavelength. trum may depend on the emission wavelength monitored, this wavelength should be specified. The polarization is usually given as r or P.

‘ihc

The corrected luminescence emission pofarization spectrum is the (fluorescence, pbosphorescence) spectrum observed when r (or P) is measured as a function of emission wavelength using a fixed and specified excitation waveiength. 4.8 Quantitazive analysis The analytical procedure used in Ium$nescence specttomerry Part III, Section 4, of thjs series of documents,

is similar

to that described

in

In fluorescence anafysis, the blank measure is p~dami~antIy due to scattering of the exciting radiation, especiallyRaman scattering_. Fluorescence from the solvent and sample cuvette as well as light scattermg in the spectrometer can also be important. In

~h#sFhQrescencc

solvent

amlysis

and sample cuvette.

the

blank

measure

is due to pbos~ore$ce~t

impurities

in the

other methods af luminescence analysis would include ~e~I~inesceace analysis, where a reaction produces bninescence radiation. A blank measure must also be wade for this method, The evaluation and assessment ments (Parts I, II and Iit].

af the analytical

result

has been dealt with in previous

docu-

5-E Geometric arr~gemeut of sample lbe luminescence measured may depend on the directions of the excitine and mirtine beams with respect to the sample. nte”angles refating to excitation and em&ion directions can be expressed by two figures, o/B where 0: = angle of incidence of the exciting beam on the plane surface of the sample, and f) = an&z between the exciting direction and observation direction. Front krface geometr;is defined as a syste; where excitationand observation are from the same face of the sample (crc90°, B
5.2 &e-filter, post-filter and self-absorption effects ?he autos does not see a po~tiou of the lminescent volume where: the excitation beam enters the sample. Ibus the exciting beam flux is reduced by absorption by the analyte and interfering impurity before it enters the voltune observed by the detection system. ‘Ibe post-filter effect arises when the exciting beara does not fill. the calI coa@etely and lminescence is absorbed by the analyte and interferixxgimpurities in the ~~n-~l~~mi~ated region facing the detectw. self-absorption effect is the reabsorption impurities within the excitation volume.

?he

AlI. three effects highly diluted.

are minimi scd if

front

surface

of iuminescence

by the analyts

geometry is used and/or

if

and interfering

the solution

is

5.3 Refraction effects ‘ibe luminescence flux emitted from the interior of a rect;mguIar sammplcreaching a photodetector Place at some distance from the sample is decreased by a factor uf approximately n2 (where n is the refractive index of the medium) compared with a medium whose refractive index is 1.0. Such effects are termed refraction effects.

5.4 Solvent and temperature effects The type of solvent and its temperature can effect the lumincscencc yield from an analytc as a result of quenching, exciplcx formation, aggregation, etc. Tempcratute effect is the term used for changes in the luminescence parameters caused by changes in temperature while solvent effects are changes caused by altering the solvent or the solvent properties (see also Section 2.2.4).

TABLE 4.2

Name

Measured

corrected

Classification luminescence

Emission spectrum

Excitation spectrum

and symbols parameters Lifetime

Em

T

Ec

T

for

Quantum yield

m

ym

c

yc

Degree of anisotropy

Polarization emission

E

PC

spectrum excitation