Luminescence

Fluorescence: short-lived excited singlet (10-9 - 10-5 s) (emission from : long-lived excited triplet, change in e- spin photo-excited species) n C ¾¾h¾1® C*1 ® C*2 long-lived

® C + hn2

(Optional) : emission from an excited species formed chemically A + B ® C* + D C* ® C + hn Low detection limit, large dynamic range, high sensitivity (and strong matrix effect)

Difference spin states (Fig. in Sec. 15A-1 or equiv) Singlet: 2e- spin are antiparallel, S = 0, diamagnetic Doublet: free radical, 2 possible spin states, S = ½ or - ½, paramagnetic Triplet: all e- spin are parallel, S = 1. The triplet excited state is less energetic than the singlet excited state.

Jablonski Energy level diagram (Fig. 15-1 or equivalent) http://micro.magnet.fsu.edu/primer/techniques/fluorescence/excitation.html for an interative demo

’ UV-visible absorption S0 ® S1 l1, lr l2 S0 ® S2 -14 -15 (10 - 10 s) S0 ® T1 forbidden

Fluorescence (excited singlet S1 to ground singlet S0) l3 (Stokes shift) l3 > lr ’ lr (= lr ) Weak absorbing t = 10-6 - 10-5 s Strong absorbing t = 10-9 - 10-7s

Phosphorescence (excited triplet to ground singlet) t = 10-4 – 10 s Singlet/Triplet transition is a significantly less probable (forbidden) event than the singlet (singlet transition)

Fig 15-3 excitation, fluorescence emission and phosphorescence emission spectra

Radiationless deactivation If this rate is faster than the photoemission rate, there is less or even no photoluminescence observed. This effect is generally reduced at low temperature and in highly viscous solvents. There are various pathways.

Vibration Relaxation (kv) In solutions, excess vibrational energy is lost because of collisions between molecules (t < 10-12s) Since the lifetime of a vibrationally excited molecule (10-12 s or less) is shorter than that of an electronically excited one (10-9 - 10-5 s), fluorescence always involves a transition from the lowest vibrational level of an excited electronic state.

Dissociation occurs (kd) if the vibration energy is sufficiently high that a covalent bond is broken.

Internal conversion (kic)

Chem 316/P. Li/lumin/P.1 This is an intermolecular process by which a molecule passes to a lower energy excited state without photoemission.

(i.e. S2 ® S1). This occurs when there is an overlap in vibrational energy levels in two electronic states (which is more prevalent when the electronic states are more closely spaced as in transition metal complexes). So although the molecules can be excited at different ls (e.g. 250 and 350 nm for quinine), they fluoresce at the same l (e.g. 450 nm for quinine) (R1* Fig. 10-4 or Fig. 15-2)

Interconversion also occurs from the first excited state to the ground state (i.e. S1 ® So). In this case, the molecule will not fluoresce. This situation prevails in aliphatic cpls. - Interconversion may also result in pre-dissociation kpd when an e moves from a higher excited state to an upper vibrational level of a lower excited state in which the vibrational energy is great enough to break a covalent bond.

External Conversion or Collisional quenching (kec) This is deactivation of an excited state due to interaction (energy transfer) between the molecule and the solvent or other solutes (e.g. halide ions, or quencher molecules). This interaction is less at lower temperatures and with more viscous solvents. This is more detrimental in phosphorescence because of longer lifetime (of the triplet state). Hence, phosphorescence is best carried out at low temperatures (in liq N2) or when the molecules are absorbed on solid surfaces.

Intersystem crossing (kisc) This is a process in which the spin of an excited e- is reversed, and a change in spin multiplicity of the molecule results. The occurrence is more probable if the vibrational levels of the two states (singlet and triplet) overlap, and if the molecule contains heavy atoms (e.g. I or Br), which promotes spin-orbital interaction. Intersystem crossing is also enhanced by the presence of paramagnetic species (e.g. O2 , transition-metal ions) in solution.

Luminescence Intensity

Since IF is proportional to the radiant intensity of the excitation source absorbed, -Îbc IF = K’ (Po - P) = K’Po (1 - 10 ) and K’= k fF where k is a constant accounting for the efficiency of collecting and detecting the fluorescence emission.

Quantum yield (or efficiency) fF The ratio of number kf to molecules that luminesce to the total number of excited molecules can be expressed as fF = kf / [kf + kd + (kic + kpd) + kec + kisc] kf, kd, and kpd mainly depend on the chemistry structure; the other values are strongly influenced by the environment.

Effect of chemical structure on f Various molecular orbitals are shown in Fig. 14-3 or equiv. (1) p* ® p (10-9 - 10-7s): greater molar absorptivity, greater energy difference between singlet and triplet (low ki), e.g.quinoline. (2) p* ® n (10-7 - 10-5s): lower molar absorptivity, lower energy difference between singlet and triplet that allows

intersystem crossing (large ki), e.g. pyridine. (3) s* ® s (150 - 250 nm) transitions seldom occur because of bond rupture.

Effect of structural rigidity on f

A lack of rigidity causes an enhanced internal conversion (at higher kic)

Chem 316/P. Li/lumin/P.2 While fluorene fluoresces, biphenyl does not. The fluorescent intensity of 8-hydroxyquinoline is much less than that of the zinc complex(Figure in section 15A-4).

Effect of temperature and solvent on f f decreases with increasing T (high kic) (see Figure), decreasing the solvent viscosity (high kec) (see Figure), and increasing the number of heavy atoms in solvents and other solutes (e.g. CBr4) (high ki)

Effect of pH on f Aniline is more resonance-stabilized than anilinium ion, and so the neutral species present in high pH solution has a higher f. Fluorescein has higher f at a higher pH.

Effect of Dissolved O2 on f Photochemical oxidation will occur. Intersystem crossing is promoted by paramagnetic O2 (high ki). The phenomenon of decrease in f due to the presence of a substance is called quenching.

Effect of concentration on f This is employed in quantitative chemical analysis. -Îbc Since IF = K’ Po (1-10 ) 2 3 ' é (2.303Îbc) (2.303Î bc) ù = K Po ê2.302Îbc - + -...ú ë 2! 3! û

= 2.3 K’Po Îbc if 2.3Îbc = A < 0.05

If A > 0.05, the linearity of F & c is lost. This and two other effects (self-quenching and self-absorption) cause a negative deviation from linearity (or appearance of a maximum)

At higher concentrations, self-quenching occurs (Œhigher collisional rate or kec) and self-absorption occurs (Œ emission being absorbed by other molecules)

Fluorescent intensity F depends on lamp output Po. So greater SNR can be obtained with an intense source (e.g. ).

Emission spectra, which depend on lamp characteristics (i.e. P0), transducers (or detector) response and monochromator, vary in different instrument. In contrast, in absorbance method, increase Po will increase P, giving similar absorbance logP0/P.

Instruments In general, the instrument for fluorescence and phosphorescence measurement (Fig. 15-4) consists of a source, excitation and emission l selectors—filter (absorption or interference) in a fluorometer (Fig. 15-6) or or monochromator (grating or prism) in a spectrofluorometer Fig. 15-7), and photodetector (PMT). The sample and reference beams pass to a differential amplifier (op amp) to produce an output.

Sources A highly intense source is needed. Low-pressure Hg vapor lamp: produces intense lines at (254, 302, 313, 546, 578, 691, 773 nm) High-pressure Xe : produces continuum (300 - 1300 nm). (Fig. 6-18 or equiv.) Chem 316/P. Li/lumin/P.3 Laser: small excitation volume, intense, highly monochromatic and collimated.

Wavelength selectors and radiation transducers are the same as in atomic spectroscopy.

Cells Made of glass or silica. -5 Instrument standardization - 10 M quinine sulfate (lex: 350 nm lem: 450 nm)

Phosphorimeter (IP=2.3 K’Po Îbc) This requires two components added on a fluorometer or spectrofluorometer(Fig. 15-9). (1) Rotating device: this will alternately irradiate the sample and,measure the phosphorescence intensity after a suitable time delay in order to differentiate between long lived phosphorescence emission and short-lived fluorometer emission.

(2) Dewar flask with quartz window: this is filled with liquid N2 to prevent collisional deactivation. The sample is usually dissolved in solvents such as a mixture of diethylether, pentane and ethanol.

Fluorometeric applications Organic species Any organic compounds (e.g. enzymes, vitamins, drugs, labelled DNA) that fluoresce are amenable to quantitation by fluorescence. Inorganic Cations Mostly amenable to non-transition colorless cations (e.g. Al3+, Cd2+) which forms fluorescent chelates with fluorometeric reagents or organic ligands (e.g. 8-hydroxyquinoline). Transition-metal ions chelates do not usually fluoresce because 1. The ions are paramagnetic, which enhances intersystem crossing. 2. The complexes are characterized by many closely spaced energy levels, which enhances internal conversion.

Photoluminescence provide a sensitive detection method for LC and CE.

Exercises 15-1, 15-3 to 15-9;

Chem 316/P. Li/lumin/P.4