2 MOLECULAR FLUORESCENCE 2.1 Introduction Spectroscopic techniques can be classified in two different ways: 
by the species interacting with the radiation – atomic or molecular 
by the type of interaction being measured – absorption or emission CLASS EXERCISE 2.1 Complete the following table by classifying the spectroscopic techniques you are familiar with: 
AAS 
flame emission 
UV‐VIS 
IR 
ICP Atomic Molecular Absorption Emission 2.2 Interaction with radiation The absorption of energy by electrons in molecules produces an excited state, and the source of the energy can be thermal, chemical or radiative. The latter is the basis for UV/VIS absorption spectroscopy, which you are already familiar with. The lifetime of the excited state molecule is very brief, typically nanoseconds, except in some special instances. For most excited state molecules, the pathway back to the ground state will be via collisions, and consequent loss of energy, with other molecules, especially solvent molecules where the absorbing species is in solution. This is known as non‐radiative relaxation, and simply means that the molecules returns to the ground state (relaxes) loses its extra energy without the emission of any radiation. If the molecule is in the gas phase, where collisions are relatively rare, or if it possesses certain structural features, which will be outlined later, it has the chance of losing the energy by another pathway, which does involve emission of radiation. The emission of ultraviolet or visible radiation by excited state molecules is called luminescence. There are various forms of luminescence, based on the source of the initial excitation energy: 
photoluminescence – radiation 
thermoluminescence – heat 
chemiluminescence – chemical reaction 
bioluminescence – within living organisms 
triboluminescence – from the fracture of crystals 2. Molecular Fluorescence It is only photoluminescence that we will consider here. Photoluminescence has two forms, based on the lifetime of the excited state. Fluorescence is the more common, and analytically useful form, where the emission occurs within nanoseconds of the excitation. Phosphorescence can occur for minutes after the excitation energy is removed. We will concentrate on fluorescence here. 2.3 The nature of fluorescence The term fluorescence is used in a number of analytical techniques, and basically refers to the emission of radiation, where the energy emitted is less than that absorbed. Fluorescence is observed with molecules, which is what this chapter is about, and involves the same sort of species as for UV/VIS absorption. It is also observed with atoms, in a technique not dissimilar to atomic emission, and X‐ray fluorescence, where the X‐ray region of the electromagnetic spectrum is involved. CLASS EXERCISE 2.2 How does this definition of fluorescence translate to the relationship between wavelengths? Fluorescence is emission of radiation, where the wavelength emitted is SHORTER/LONGER than that absorbed. Why is the emitted energy less? When a molecule absorbs UV/VIS energy, the excited state, like the ground state, has a large number of vibrational states associated with it. In the ground state, it is these vibrational states that provide the excited states for infrared absorption spectra. In the excited state, it provides the reason that UV/VIS absorption peals are so broad. There are many very close together states which are part of the absorption process. This is shown in Figure 2.1(a). Depending on the conditions (temperature etc) and the compound, more or less of the excited state molecules will follow the non‐emission pathway (2.1b). Those which can emit the radiation will do so from the bottom “rung” of the excited state, and will drop back to various vibrational levels in the ground state. Therefore, we see that the fluorescence energy drops are on average less than the absorption rises. Excited State (a) (b)
(c) Ground State FIGURE 2.1 Energy transitions in molecular fluorescence (a) absorption (b) non‐radiative relaxation (c) fluorescence AIT 2.2 2
2. Molecular Fluorescence
e Figure 2
2.2 shows th
he absorptio
on and fluorrescence speectra of two
o different compounds, c
and clearlyy shows th
he longer waavelengths fo
or the latter spectra. FIGURE 2
2.2 Absorptio
on and emissiion spectra fo
or anthracenee (A – absorpttion, C – fluorrescence) and
d quinine (B –– absorptio
on, D – fluoresscence) (from
m GG Guilbart, Practical Fluo
orescence, Deekker) no situation, but rather a scale from 0
0% to 100%.. In realityy, whether a compound fluoresces iss not a yes/n
The greaat majority of o compounds fall into the t very low
w to zero end of the scaale, but some do exhibitt some deegree of fluorescence, as shown in Figgure 2.3. naphthalenee quininee
ch
hlorophyll fluorescein 10% 55%
30%
90% 0% F 10
00% F
100% NRR 0% NRR FIGURE 2
2.3 Fluorescen
nce scale (F m
means fluoresccence, NRR meeans non‐radiative relaxatio
on) molecule fluo
oresce stron
ngly? 2.4 Whaat makes a m
First and
d foremost, ffor a species to fluorescee strongly, it must first ab
bsorb stronggly: if it canno
ot get to thee excited sstate, then itt can’t comee down from it! Therefo
ore, any subsstance that d
does not absorb stronglyy in the U
UV/VIS region – for reassons you sho
ould be awaare of from earlier subjeects – have no hope off fluoresciing. AIT 2.3 2. Molecular Fluorescence However, not all absorbing species will fluoresce. The various coloured inorganic ions, such as copper (II) and permanganate, do not. Benzene fluoresces weakly. It is not obvious what makes some inorganic species, such as Ce3+ fluoresce, when others that are similar, eg Ce4+, do not. None of the important ions – cation or anion – fluoresce, but as will be seen the Applications section, they can be reacted to form a species that does. For organic compounds, it is necessary that the absorbing part of the molecule be structurally rigid, so fused aromatic rings, where the rings are joined at two points will lead to greater fluorescence than a similar molecule where the rings are joined by a single bond, as shown in Figure 2.4. Naphthalene (10% fluorescence) Phenylbenzene (<1% fluorescence) FIGURE 2.4 Effect of rigid structure on fluorescence 2.5 Non‐structural factors affecting fluorescent intensity Matrix Matrix conditions can substantially affect the intensity of a fluorescent solution. This process is known as quenching, and anything that causes a decrease in fluorescent intensity is known as a quenching agent. Species or conditions include:  pH – acids or bases when in the ionised form may be much less fluorescent, eg benzeneamine is not fluorescent at all in acidic conditions 
temperature – higher temperatures reduce fluorescent intensity 
heavy atoms ‐ whether present in the solvent or in the matrix, heavy elements such as Br or I cause a decrease in fluorescence of the analyte 
dissolved oxygen – this reduces fluorescence 
ligands – complexes of metal ions with rigid aromatic ligands produce fluorescence, but if the sample includes other ligands (eg F‐ and EDTA) which compete with the fluorescent ligand, a loss of intensity will occur Spectroscopic As with UV/VIS absorption spectroscopy, the wavelength chosen for measurement will make a great difference to the intensity. As with the absorption technique, the recommended wavelength is at the top of the peak. However, in the case of fluorescence, there are two radiation processes involved, and each affects the fluorescent intensity. The wavelength of radiation shining on the sample which causes the excitation determines how much light is absorbed, and therefore how many excited state species are generated. The greater the absorption, the greater the fluorescence, so it is logical to choose the wavelength of maximum absorption. This is shown very clearly in Figure 2.5. The fluorescent radiation that is generated occurs over a range of wavelengths, and the wavelength of maximum intensity should be chosen, unless there is a close proximity (< 30 nm) between this and the first wavelength. The two wavelengths are usually called the excitation wavelength and the emission wavelength. Typically the latter will be 40‐60 nm longer the former. AIT 2.4 2
2. Molecular Fluorescence
e FIGURE 2
2.5 Multiple scans of thee fluorescencee spectrum off perylene (sttructure show
wn), recorded at excitation
n waveleng
gths with 6 nm intervals from fr
300 to 456 4 nm (from
m Christian & O’Reilly, Instrrumental Ana
alysis, Allyn & &
Bacon) CLASS EXEERCISE 2.3 Using th
he spectra in
n Figure 2.2
2 and Figuree 2.5, choosee the best combination c
of wavelen
ngths for (a)) quinine ((b) anthracene and (c) peerylene. Compoun
nd Excita
ation Wavellength ngth Emissiion Wavelen
quinine anthraceene perylenee n 2.6 Instrumentation
n for fluoresscence sharees much with UV/VIS ab
bsorption insstruments, because b
it iss The instrumentation
hat is involveed. Howeve
er, there are a number o
of significantt after all the same reegion of the spectrum th
2.6 shows the general co
omponents o
of a fluoresce
ence instrum
ment. differencces. Figure 2
Collim
mator
Sam
mple Excitaation  Radiaation Sou
urce C
Cell Seleector Collim
mator
Read
dout
Deteector
Emission  ector Sele
FIGURE 2
2.6 Schematicc diagram of a
a fluorescencee instrument AIT 2.5 2. Molecular Fluorescence Radiation source In an absorption instrument, this is a low intensity lamp; in a fluorescence instrument, the intensity must be much greater because of the need to generate as many excited state species as possible. The most common lamps are Xe‐arc or Hg‐arc: each produces intense radiation across the UV/VIS range. Collimators These are focussing lens to maximise the intensity of the beam shining on the sample and into the detector. Wavelength selectors As mentioned above, there are two wavelengths that must be selected to maximise emission intensity. You might think that it would be best if all wavelengths, even those not strongly absorbed, shone on the sample to maximise absorption. However, if this was done, the photodecomposition of the analyte, which occurs anyway, would be severe and a problem. As with absorption instruments, filter and monochromator wavelength selectors exist. In absorption instruments, filter photometers are little different in their quantitative performance than expensive monochromator‐based instruments. In fluorescence, the use of filters in one or both positions (almost always the excitation) actually increases sensitivity, because more radiation hits the sample, and more hits the detector. Of course, the double‐filter instruments cannot record an emission spectrum. With a double‐filter system, the transmission spectra of the two filters cannot overlap, otherwise stray light problems will occur, even at 90. Sample cell Similar requirements apply here in terms of cell material as with absorption instruments: silica, not quartz (which fluoresces) for the UV, glass for the visible. However, the cell must be polished all around (all four sides if square), because the emission intensity is measured at 90 to the excitation beam. This is to avoid radiation form the lamp hitting the detector (stray light). Fluorescent radiation is generated in all directions, so it doesn’t affect its intensity wherever the detector is placed. Detector A photomultiplier tube, as with absorption instruments. 2.7 Practical aspects Choosing the analytical wavelengths Typically the two wavelengths needed for maximum sensitivity are about 50 nm apart, the second being higher. Selecting them is a bit like the chicken and the egg story: unless you get one wavelength right, you can’t correctly work out the other. There is a simple method for selecting them: the wavelength of exciting radiation which causes the most fluorescence is equal to the wavelength of maximum absorption, easily measured on a UV/VIS absorption instrument. Choose the filter closest to this value for the excitation selector. How to determine the best emission wavelength will depend on the type of instrument. If the second wavelength selector is a monochromator, then a scan (manual or machine) will find the highest emission. If it is a filter, then add 50 nm onto the absorption maximum and look for the filter closest. If this doesn’t work, try the next longest wavelength filter until sufficient response is achieved. Calibration range As with absorption measurements, getting the linear range for the species can be trial and error, if you have no idea where to start. However, in fluorescence, there is another problem, which is illustrated in Figure 2.7. AIT 2.6 2. Molecular Fluorescence Intensity
Concentration
FIGURE 2.7 The response curve for fluorescence This behaviour, where at high concentrations, the intensity begins to decrease is known as self‐
quenching. It is due to analyte molecules being able to absorb some of the radiation that others have emitted because of the partial overlap of the absorption and emission spectra. The newly created excited state molecule may not fluoresce, or if it does, the photon may not travel in the same direction as the initial one. CLASS EXERCISE 2.4 Why is the self‐quenching behaviour a problem? The way to check where in the response curve your solution actually is, is to do a quick, approximate 1:1 dilution with solvent, and check the intensity. If it has decreased, then your sample is in the linear region; if it increases, it is in the self‐quenching region, and require a large dilution to get it into the working range. 2.8 Applications of fluorescence A small, but important, selection of organic analytes exist to make fluorescence a viable technique. Of these, the most important are in the pharmaceutical area (eg Vitamin A, barbiturates, amphetamines, barbiturates) and air pollution monitoring (eg the polycyclic aromatic hydrocarbons (PAHs) such as benzo[]pyrene – see Figure 2.8), which are the highly carcinogenic by‐product of combustion of fossil fuels). Fluorescence detectors are also important in HPLC instruments. AIT FIGURE 2.8 Benzo[]pyrene, a polycyclic aromatic hydrocarbon 2.7 2. Molecular Fluorescence As mentioned earlier, few if any of the important inorganic ions fluoresce – none of them cations. There are methods that have been developed to analyse metals and even some anions using complex formation with fluorescent ligands. However, advances in other techniques (eg ICP, ion chromatography) have meant these applications of fluorescence are not often used. 2.9 Advantages and disadvantages The technique with which fluorescence should be compared is UV/VIS absorption, because of the similarity of analytes. Therefore, the following points are specifically using that comparison. Advantages 



greater sensitivity – the intensity for medium‐strong fluorescing species means that the lower limit of the working range is in the 100‐500 ug/L range, compared to 1‐10 mg/L for absorption measurements; typically the top end of the working range for fluorescence is in the middle of the linear region for absorption greater linear region – typically 100 times, compared to 10 times maximum for absorption measurements selectivity – this can be both an advantage or disadvantage (see below); the smaller range of species that fluoresce mean that some interfering species in absorption measurements do not fluoresce and are therefore not a problem; also, the necessity to choose two wavelengths increases the chance of separating similar species cheapness – because the double‐filter fluorometer is actually more sensitive than its monochromator‐equivalent, you get a better instrument for quantitative analysis at a fraction of the price; in absorption, filter photometers do not have any performance advantage except portability Disadvantages 

limited species availability – the advantage of selectivity is also a problem, in that your analyte may not fluoresce one extra step – the rough 1:1 dilution of all samples takes a little longer, and doesn’t have an equivalent in absorption What You Need To Be Able To Do 
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AIT define important terminology explain how excited state species lose energy explain why fluorescence is at a higher wavelength than absorption identify compounds that will fluoresce strongly draw a schematic diagram, showing the components of a fluorimeter explain the function of each component describe practical aspects of setting up and performing a fluorometric analysis list factors that cause quenching explain the significance of self‐quenching 2.8 2. Molecular Fluorescence Revision Questions 1. How is fluorescence different to emission? 2. How do most compounds lose their absorbed energy when going back to the ground state? 3. Rank the following compounds in order of increasing fluorescence. I. III. 4. 5. 6. 7. 8. II. IV. Intensity
0 75 304 1526
268 151 896 975
Would you recommend the purchase of a fluorimeter for a general laboratory? Explain your answer. Answers on following page. AIT What is the meaning of the terms quenching and self‐quenching? Why is the wavelength of maximum absorption shorter than that of maximum fluorescence? Why are two wavelength selectors necessary in fluorescence measurements? How do you determine the correct wavelengths for measuring fluorescence with a (a) double‐
filter and (b) filter‐monochromator instrument? Determine the concentration of samples A & B, given the data below. Solution Blank 0.5 mg/L 2 mg/L 10 mg/L Sample A Sample A (diluted approx. 1:1) Sample B Sample B (diluted approx. 1:1)
9. 2.9 2. Molecular Fluorescence Answer Guide Where the answer can be found directly in your notes, a reference to them will be provided. 1.
The energy emitted is less than that absorbed. 2.
Small amounts in numerous collisions (non‐radiative relaxation) 3.
III (least), II, IV, I (most) 4.
quenching – 2.4, self‐quenching – 2.7 5.
Higher energy means lower wavelength 6.
section 2.6 7.
(a) record emission spectra at different excitation wavelength ; find highest peak in emission spectra and excitation wavelength giving highest emission intensity (b) recording UV‐VIS absorption spectrum to find excitation wavelength; add 40 nm for emission filter 8.
A – 1.76 mg/L, B cannot be determined as it is in the self‐quenching region, indicated by diluted intensity increasing 9.
No, its applications (analytes) are not sufficiently wide to be useful in a general laboratory. AIT 2.10