5

MOLECULAR FLUORESCENCE

5.1 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 5.1 Complete the following table by classifying the spectroscopic techniques you are familiar with: AAS flame emission UV-VIS IR NMR ICP Atomic Absorption Molecular

Emission

5.2 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

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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. 5.3 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 5.2 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 5.1(a). Depending on the conditions (temperature etc) and the compound, more or less of the excited state molecules will follow the non-emission pathway (5.1b). 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 5.1 Energy transitions in molecular fluorescence (a) absorption (b) non-radiative relaxation (c) fluorescence

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Figure 5.2 shows the absorption and fluorescence spectra of two different compounds, and clearly shows the longer wavelengths for the latter spectra.

FIGURE 5.2 Absorption and emission spectra for anthracene (A absorption, C fluorescence) and quinine (B absorption, D fluorescence) (from GG Guilbart, Practical Fluorescence, Dekker)

In reality, whether a compound fluoresces is not a yes/no situation, but rather a scale from 0% to 100%. The great majority of compounds fall into the very low to zero end of the scale, but some do exhibit some degree of fluorescence, as shown in Figure 5.3.
naphthalene 10% chlorophyll 30% quinine 55% fluorescein 90%

0% F 100% NRR
FIGURE 5.3 Fluorescence scale (F means fluorescence, NRR means non-radiative relaxation)

100% F 0% NRR

5.4 What makes a molecule fluoresce strongly? First and foremost, for a species to fluoresce strongly, it must first absorb strongly: if it cannot get to the excited state, then it cant come down from it! Therefore, any substance that does not absorb strongly in the UV/VIS region for reasons you should be aware of from earlier subjects have no hope of fluorescing. 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. AIT 5.3

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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 5.4.

Naphthalene (10% fluorescence)

Phenylbenzene (<1% fluorescence)

FIGURE 5.4 Effect of rigid structure on fluorescence

It has been shown that rigidity in the bulk material will also increase fluorescence. Incorporating a fluorescent substance into a plastic film increases its intensity over a solution of similar concentration. Likewise, lower temperatures will increase sensitivity.

5.5 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 5.5. 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.

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FIGURE 5.5 Multiple scans of the fluorescence spectrum of perylene (structure shown), recorded at excitation wavelengths with 6 nm intervals from 300 to 456 nm (from Christian & OReilly, Instrumental Analysis, Allyn & Bacon)

CLASS EXERCISE 5.3 Using the spectra in Figure 5.2 and Figure 5.5, choose the best combination of wavelengths for (a) quinine (b) anthracene and (c) perylene. Compound quinine anthracene perylene 5.6 Instrumentation The instrumentation for fluorescence shares much with UV/VIS absorption instruments, because it is after all the same region of the spectrum that is involved. However, there are a number of significant differences. Figure 5.6 shows the general components of a fluorescence instrument.
Radiation Source Excitation Selector Sample Cell

Excitation Wavelength

Emission Wavelength

Collimator

Collimator

Readout

Detector

Emission Selector

FIGURE 5.6 Schematic diagram of a fluorescence instrument

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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 doublefilter 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 doesnt affect its intensity wherever the detector is placed. Detector A photomultiplier tube, as with absorption instruments. 5.7 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 cant 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 doesnt work, try the next longest wavelength filter until sufficient response is achieved.

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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 5.7.

Intensity

Concentration

FIGURE 5.7 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 5.4 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 selfquenching region, and require a large dilution to get it into the working range. 5.8 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 5.8), which are

FIGURE 5.8 Benzo[]pyrene, a polycyclic aromatic hydrocarbon

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the highly carcinogenic by-product of combustion of fossil fuels). Fluorescence detectors are also important in HPLC instruments. 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, such as those shown in Figure 5.9. However, advances in other techniques (eg ICP, ion chromatography) have meant these applications of fluorescence are not often used. (a) (b)
OH NaO 3S HO

N=N

N OH
FIGURE 5.9 Common fluorometric reagents (a) 8-hydroxyquinoline (b) Alizarin garnet R

5.9 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 doesnt have an equivalent in absorption

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What You Need To Be Able To Do 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 describe analytical methods that allow fluorometric analysis of non-fluorescent cations and anions compare fluorescence and UV-VIS absorption for quantitative analysis

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.

II.

III. 4. 5. 6. 7.

IV. 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?

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8.

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) Intensity 0 75 304 1526 268 151 896 975

9.

Would you recommend the purchase of a fluorimeter for a general laboratory? Explain your answer.

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