Fluorescence Lecture 1 PDF

Volumetric Quantitative Gravimetric Electrochemistry Analytical Chemistry Qualitative Instrumental Spectroscopy Chromatography Spectroscopy is the interaction of EMR with matter; p...

Volumetric Quantitative Gravimetric Electrochemistry Analytical Chemistry Qualitative Instrumental Spectroscopy Chromatography Spectroscopy is the interaction of EMR with matter; part of EMR is reflected Ir, part is refracted If, part is scattered Is, part is absorbed Ia and part is transmitted It  A process that occurs when Luminescence A chemical reaction which photons of EMR are spectroscopy yields an electronically absorbed by molecules, excited product, which emits raising them to some excited light on returning to ground state, and then, on returning state to ground state, the molecules emit radiation Photoluminescence Chemiluminescence The life time of excited state is very short (10-9 -10-6 sec) The life time of excited state is longer (10-3 - 100 sec) Fluorescence Phosphorescence Resonance Stockes shift fluorescence  Theory of molecular fluorescence spectroscopy  Before excitation, molecules are mostly in the lowest vibrational level of the ground electronic state.  Upon absorption of energy (10-15 sec), the excited electron enters a higher vibrational level in the excited state.  An excited molecule can return to the ground state by different relaxation (deactivation) processes Radiationless Radiational (Non-radiative) (Radiative) deactivation deactivation The favored route to the ground state is the one that minimizes the life time of the excited state  Radiationless deactivation Vibrational Internal relaxation conversion External Intersystem conversion crossing  Radiative deactivation Fluorescence Phosphorescence Jablonski energy diagram  Vibrational relaxation:  Excited molecules normally relax rapidly to the lowest vibrational level of the excited electronic state.  Occurs in solution and involves transfer of energy to neighbouring molecules due to collisions between excited molecules and those of solvent resulting in slight increase in solvent temp.  This process is very rapid in solution (10-12 - 10-10 sec).  Internal conversion:  A process by which the molecule passes from an excited electronic energy level (S2) to another excited energy level (S1).  This occurs when the lowest vibrational energy level of S2 coincide with one of the vibrational levels of S1.  Following internal conversion, the molecule loses further energy by vibrational relaxation and will relax to the lowest vibrational energy level of the lowest excited singlet electronic state before any radiation is emitted.  When the molecule has reached the lowest vibrational energy level of the lowest singlet excited electronic energy level (S1) then a number of events can take place: a) External conversion i.e. continue in non radiative deactivation (if it’s a non fluorescent molecule) or b) Radiative deactivation (fluorescence or phosphorescence) 1- The molecule can lose energy by related form of radiationless relaxation; external conversion, in which excess energy is transferred to the solvent or another component in the solution (energy is lost as heat). 2- Fluorescence:  The emission of a longer wavelength radiation by a substance as a consequence of absorption of energy from a shorter wavelength radiation  Fluorescence occurs when the electron found in an excited singlet level (S2) converted to excited singlet state but of lower energy (S1) by internal conversion then relaxes to the ground state with emission of radiation (S1-S0) 3- The molecule can undergo intersystem crossing and emit radiation equal to the energy difference between the lowest triplet energy level and the ground-state in a process known as phosphorescence (T1-S0).  The outer most electrons are usually: i) Even number ii) Paired electrons iii) With no net electron spin i.e. no net magnetic field (diamagnatic). This electronic state in which electron spin is paired is called a singlet state.  When one electron is excited to a higher energy level a singlet or a triplet state can result.  In the singlet state, the spin of the promoted electron is paired with the ground state, while still in the excited state,  In the triplet state, the electron may reverse (flip) its spin and the two electrons become unpaired and thus have the same spinning i.e. there is a magnetic field (paramagnetic).  Intersystem crossing 1- This occurs when the lowest vibrational energy level of S1 coincide with one of the vibrational levels of T1(N.B. triplet state is less energetic than the corresponding singlet state). 2- It is a process in which the spin of an excited electron is reversed (flipped) i.e. has a spin that is identical to that of electron in the ground state, 3- It is common in molecules containing heavy atoms such as I2 and Br2 also it is enhanced in presence of paramagnetic molecules such as molecular oxygen (O2)  POC Singlet state Triplet state Types Ground or excited Excited Magnetic effect Diamagnetic (Paired) Paramagnetic (Unpaired) Luminescence Fluorescence Phosphorescence Life time of excited state 10-9 -10-6 sec 10-3 -100 sec Electron transition for More probable Less probable emission Since the triplet to singlet (or reverse) is a forbidden transition, meaning it is less likely to occur than the singlet-to-singlet transition, the rate of triplet to singlet is typically slower. Therefore, phosphorescence emission requires more time than fluorescence. Fluorescence excitation and emission spectra for a solution of quinine. In an emission spectrum, the excitation wavelength is held constant and the fluorescence intensity is measured as a function of the emission wavelength. In an excitation spectrum, the emission wavelength is held constant and the fluorescence intensity is measured as a function of the excitation wavelength. The excitation spectrum closely resembles an absorption spectrum since the fluorescence intensity is usually proportional to the absorbance of the molecule  Excitation and emission spectra  A fluorescent species has 3 Spectra  A sample’s excitation spectrum is nearly identical to its absorption spectrum. If the 2 spectra are plotted on same chart, Stokes shift is apparent and have a mirror image relation to each other Resonance fluorescence: Less commonly (occur in atoms), the absorbed and emitted radiations have the same energy (same wavelength). The Stokes shift: the emitted radiation has longer wavelength i.e. less energy than the absorbed radiation. This is due to the fact that some of the energy of the excited fluorophore is lost through molecular vibrations that occur during the brief lifetime of the molecule’s excited state. This energy is dissipated as heat to surrounding solvent molecules as they collide with the excited fluorophore.  Quantum yield or quantum efficiency Fluorescence, phosphorescence and external conversion are competing processes. Since molecules return to their ground state by the fastest mechanism, fluorescence is only observed if it is a more efficient means of relaxation than the combination of external conversion and vibrational relaxation.  A quantitative expression of the efficiency of fluorescence is the quantum yield (quantum efficiency), Φ , (ratio of no. of molecules which fluoresce to total no. of excited molecules or ratio of photons emitted to that absorbed).  Quantum yields range from (1) when every molecule in an excited state undergoes fluorescence, to (0) when fluorescence does not occur. The intensity of fluorescence increases with:  an increase in quantum efficiency  incident power of the excitation source  concentration of the fluorescent species.  CONCENTRATION AND FLUORESCENCE INTENSITY  The power of the fluorescent radiation F is proportional to the radiant power of the excitation beam that is absorbed by the system. F α Ia → F = K Ia (where Ia = I0 – I) F = K (I0 – I)………………(1) where K depends upon the quantum efficiency of the fluorescence process.  To relate F with the concentration c of the fluorescence particle we write Beer's law in the form:  A = Log I0/I = εbc → I0/I = 10εbc  (I0/I)-1 = 10-εbc  I/I0 = 10-εbc  I = I0 * 10-εbc…………………(2) F = K (I0 – I0 * 10-εbc) F = K I0(1 - 10-εbc)  provided εbc = A < 0.05, the exponential term of the equation will be:  F=2.303 * K * I0 * εbc  F = K’ * C  A plot of fluorescent power of a solution versus concentration of the emitting species should be linear at low concentration.  When the concentration becomes greater enough so that the absorbance is larger than 0.05, linearity is lost. F For a concentration above c1 the calibration curve is no c1 longer linear. Conc. of fluorescing species The departure from linearity at high concentration is due to: a) Self-absorption: this occurs when the wavelength of emission overlaps an absorption peak then some of the emitted radiation will be absorbed by the molecules in solution and decrease in fluorescence takes place. b- Self-quenching: it results from the collision of the excited molecules→ external conversion  Spectrofluorimetric methods are among some of the most sensitive analytical methods available and their detection limits are lower than those of absorption spectrophotometry…why?  The fluorescence signal is directly proportional to the intensity of the excitation source. We can increase the intensity of the excitation source and gain a subsequent increase in the fluorescence signal—potentially leading to greater sensitivity.  Spectrophotometry, on the other hand, is an absorption technique. Absorption depends on the ratio of incident to passed light: A = Log I0/I so simply increasing I0 also increases I. Thus the ratio does not change. As a result, the sensitivity of fluorescence can be increased by increasing I0 but that of absorbance does not change.  Many materials are fluorescent, including:  Highlighter pens.  Our teeth.  Ripe bananas fluoresce bright blue under UV light, due to one of the products of chlorophyll degradation.  Fluorite (CaF2: calcium fluoride), it has a bright blue color under UV light.  When exposed to UV light, the fluorescence of human teeth gives them the quality of vitality and used by dentists for the early diagnosis of dental caries.  A TikToK video about a woman with false teeth and when she shined an ultraviolet light on her smile some of her teeth shone, and some didn't.  That's because the material used in false teeth reacts differently and they appear much darker than real ones, so the teeth that have been replaced will still leave a gap in your smile.  This means that in a nightclub if you've had a front tooth replaced when you smile at someone they will definitely be able to tell