MOLECULAR FLUORESCENCE SPECTROSCOPY Molecular absorption spectrophotometry Absorption of

MOLECULAR FLUORESCENCE SPECTROSCOPY

Molecular absorption spectrophotometry Absorption of EMR by molecules X-ray spectroscopy IR absorption spectroscopy Nuclear magnetic resonance spectroscopy (NMR) • UV-Vis spectrophotometry • • Atomic absorption spectrophotometry Measures absorption of EMR by atoms Flame AAS atomization Graphite AAS Uses external radiation for excitation Molecular emission spectrophotometry Photoluminescence: • Fluorescence spectrophotometry • phosphorescence spectrophotometry Atomic Emission spectroscopy • Measures emission of EMR by atoms • Uses flame for atomization & excitation

• Fluorescence is a form of photoluminescence; and this later is a type of luminescence that occurs when certain molecules are excited by electromagnetic radiation and as a consequence remission of radiation either of the same wavelength or longer one takes place. The two most common photoluminescence are fluorescence and phosphorescence which are produced by different mechanisms. • Fluorescence is distinguished from phosphorescence by the lifetime of the excited state, with fluorescence the excited state ceases immediately after irradiation is discontinued, (10 -7 s), while phosphorescence continued for a detectable time (100 s).

Theory of molecular fluorescence • An excited molecule can return to its ground state by combination of several mechanistic steps. Deactivation or relaxation processes can be classified to radiative and nonradiative processes.

Jablonski Diagram: showing the phenomena of fluorescence and phosphorescence

• At the ground state, the molecular orbitals are occupied by two electrons. the spins of the two electrons in the same orbital must be antiparallel. This implies that the total spin, S, of the molecule in the ground state is zero [1⁄2 + ( 1⁄2)]. • This energy state is called “singlet state” and is labeled as S 0. • The electron spins in the excited state achieved by absorption of radiation may either be parallel or antiparallel. Accordingly, this may be a triplet (parallel) or a singlet (antiparallel) state

The deactivation processes can be broadly categorized into two groups: 1) Nonradiative deactivation 2)Radiative deactivation

Radiationless deactivation: 1 -Vibrational relaxation (VR) Conversion of the excited electron from the highest energy sublevel to the lowest energy sublevel in the same main energy level. 2 -Internal conversion (IC): It is intermolecular processes by which a molecular passes from an electronic excited energy level (S 2) to another lower excited energy level (S 1). 3 -External conversion (EC): It is deactivation of an excited electronic state which involve interaction and energy transfer between the excited molecules and the solvent or other solutes.

4 - Intersystem crossing (ISC) is a process in which the spin of an excited electron is reversed. The probability of this transition is enhanced if the lowest vibrational energy level of the lowest excited singlet state is almost identical in its energy to that of the triplet excited state. • ISC is common in molecules containing heavy atoms such as iodine and bromine, also it is enhanced in presence of paramagnetic molecules such as molecular oxygen.

Radiative deactivation: 1 -Fluorescence Transition from S 2 or S 1 to the ground singlet state (S 0) occurs with loss of energy in the form of EMR (emission of photons) is termed fluorescence (S 1 or S 2 to S 0). 2 -Phosphorescence occurs when an electron in an excited triplet state relaxes to the ground singlet state while emitting radiation (T 1 to S 0).

Excitation and emission spectra: If the intensity of emitted light (fluorescence) at a fixed wavelength ( emission) is plotted as a function of wavelength of radiation used to excite a molecule, an excitation spectrum will result. On the other hand, if the intensity of emitted radiation (fluorescence) is plotted versus wavelength, an emission spectrum is obtained. In this case, the sample is irradiated with monochromatic radiation of certain wavelength ( excitation) and a scan of the wavelength of emitted radiation is recorded.

If both of the excitation and emission spectra of a compound are plotted on the same chart, the following will be observed: 1 displacement of emission band to longer wavelength (Stock’s shift). 2 - excitation and emission spectra bear a mirror image relationship to each other as shown in the following figure. Excitation and emission spectra

Quantum yield ( ): The quantum yield or quantum efficiency ( ) for a fluorescent process is the ratio of the number of molecules that fluoresce to the total number of excited molecules OR the ratio of number of photons emitted to that absorbed. For a highly fluorescent molecule approachs unity ( = 1), while for a nonfluorescent molecule =0. Quantitative fluorimetry: F = 2. 3 K bc I 0 F = K` c A plot of fluorescence intensity versus concentration is linear at low concentration. When the concentration becomes high enough, A > 0. 05 linearity is lost.

At high concentration, two main factors are responsible for deviation from linearity: 1 -Self-absorption: this occurs when the wavelength of emission overlaps with an absorption peak. Then, some of the emitted radiation will be absorbed by molecules in solution and a decrease in fluorescence takes place. 2 - Self-quenching: it results from the collision of the excited molecules.

Factors affecting fluorescence: 1 - Molecular structure • The most intense and most useful fluorescent behavior is found in compounds containing aromatic functional group. Compounds containing aliphatic and alicyclic carbonyl groups or conjugated double-bond structures may also exhibit fluorescence. Absorption max: 498 nm Fluorescence max: 518 nm Fluorescein Compounds with fused ring are found to be especially fluorescent, and the extent of fluorescence is found to be directly proportional to the number of rings in the molecule

Factors affecting fluorescence: 1 - Molecular structure • The quantum yield increases with the increase of number of fused rings. The simplest heterocyclics, such as pyridine, thiophene, pyrrole and furan do not fluoresce (the lowest transition is n - * system which is rapidly converted to triplet and prevents fluorescence). • Halogen substitution especially with bromine and iodine results in a decrease in fluorescence due to intersystem crossing.

The structural rigidity in a molecule favours fluorescence Fluorescence is favored in molecules that posses rigid planer structure. Examples: 1 fluorene fluoresce much more intense than biphenyl due to rigidity furnished by methylene group in fluorene. 2 The influence of rigidity is accounted for the increase of fluorescence of certain chelating agents when they form complexes with a metal ion e. g. the fluorescent intensity of 8 -hydroxyquinoline is much increased when it forms zinc complex. On the other hand the nonrigid molecules do not fluoresce much, as these rapidly lose the absorbed energy through nonradiative means like, vibrational relaxation or even degradation.

2 - Effect of temperature and solvent: • The quantum efficiency of fluorescence by most molecules decreases with increasing temperature, as deactivation by external conversion is favored. Also a decrease in solvent viscosity leads to the same result. • Polar solvents may enhance fluorescence, while it is decreased by solvents containing heavy atoms such as carbon tetrabromide or ethyl iodide. 3 - Effect of dissolved oxygen: Being paramagnetic, dissolved oxygen decreases the fluorescence due to quenching (decrease in fluorescence intensity) The quenching happens because an excited indicator molecule has come into contact with an oxygen molecule, transferring its excess energy to the oxygen molecule in a non-radiative transfer Oxygen deficiency fluorescence Absorption of light Lack of fluorescence Oxygen

Instrumentation It is composed of the following main parts shown in the following diagram Schematic diagram of a spectrofluorimeter

1 -Source of energy Several sources have been used, the two most commonly used are: A-Mercury–arc lamp: It is a quartz lamp containing mercury vapor which upon electrical excitation emits line spectra of several definite wavelengths. It can not be used when a scan of spectrum is required. B-High pressure xenon lamp: This lamp emits a continuum of radiation throughout the UV-Vis region so it is useful when spectrum scanning is required.

2 -Wavelength selector Two filters (either absorption or interference filters can be used) or monochromators (grating type) are used; one between the source and the sample and the other between the sample and the detector. 3 -The cell: Tetragonal or cylindrical transparent, glass or quartz tubes are used (the four sides are transparent). Compare with the sample cell in the spectrophotometry. 4 -Detectors and readout meter: Photomultiplier type is used since the intensity of emitted radiation is small. Digital or analog or null point meter are used.

Important notes: • Emission of radiation by sample takes place in all directions. The emitted radiation is measured at 900 from the path of the exciting beam and at the center of the cell. • This is to minimize the error due to scattering of light from the walls of the cell and to prevent the interference from the exciting beam, which occurs at other angles. • Since a broad emission band is obtained, it is necessary to use a second wavelength selector between the sample and the detector in order to pass the most intense emitted wavelength ( emission).

Applications of fluorimetry: • Compounds which are intrinsically fluorescent are easily determined at very low concentrations by simple fluorimetric method (Direct fluorimetry). For example, phenobarbitone, quinine, emetine, adrenaline, cinchonine, reserpine vitamin A, riboflavine and other natural products. • Nonfluorescent substances can be determined after chemical reaction (Indirect fluorimetry). • Inorganic ions can be determined either by formation of fluorescent chelates upon reaction with fluorimetric reagents e. g. 8 - hydroxyquinoline (for Al 3+), benzoin (for Zn 2+) or flavanol (for Zr 3+) or by measuring the quenching of fluorescence of a fluorescent substance in the presence of some ions.