Molecular Luminescence Spectrometry - Instrumental Analysis - Lecture Slides, Slides of Chemical Instrumentation and Analysis

Download Molecular Luminescence Spectrometry - Instrumental Analysis - Lecture Slides and more Slides Chemical Instrumentation and Analysis in PDF only on Docsity! Molecular Luminescence Spectrometry • Three types of Luminescence methods are: (i) molecular fluorescence (ii) phosphorescence (iii) chemiluminescence • In each, molecules of the analyte are excited to give a species whose emission spectrum provides information for qualitative or quantitative analysis. The methods are known collectively as molecular luminescence procedures. docsity.com • Fluorescence: absorption of photon, short-lived excited state (singlet), emission of photon. • Phosphorescence: absorption of photon, long- lived excited state (triplet), emission of photon. • Chemiluminescence: no excitation source – chemical reaction provides energy to excite molecule, emission of photon. • Luminescence: High sensitivity  strong signal against a dark background. • Used as detectors for HPLC & CE. docsity.com Deactivation • Process by which an excited molecule returns to the ground state • Minimizing lifetime of electronic state is preferred (i.e., the deactivation process with the faster rate constant will predominate) Radiationless Deactivation Without emission of a photon (i.e., without radiation) docsity.com Singlet excited states Triplet excited state a. Internal Vibrational Tt +. conversion relaxation Se f i Intersystem t = crossing A Si rtd 56 Prt 2 1rittl a Internal 1ridd and 1rttdl Absorption Fluorescence Phosphorescence external 1ridd conversion Iridd [cit i 1ridl 1rttdl Ltt lee ! f Vibrational; So Y — relaxation 4. = y y round ¥ Y | 1 te Ms Ay Ae Ae Os Ng (07 Thomson Higher Education docsity.com  TERMS FROM ENERGY-LEVEL DIAGRAM Term: Absorption Effect: Excite Process: Analyte molecule absorbs photon (very fast ~ 10-14 – 10-15 s); electron is promoted to higher energy state. Slightly different wavelength  excitation into different vibrational energy levels. Term: Vibrational Relaxation Effect: Deactivate, Radiationless Process: Collisions of excited state analyte molecules with other molecules  loss of excess vibrational energy and relaxation to lower vibrational levels (within the excited electronic state) docsity.com Term: External Conversion Effect: Deactivate, Radiationless Process: Collisions of excited state analyte molecules with other molecules  molecule relaxes to the ground state without emission of a photon. Term: Phosphorescence Effect: Deactivate, Emission of h Process: Emission of a photon via a triplet to single transition (long–lived excited state ~ 10-4 – 101s) docsity.com Quantum Yield The quantum yield or quantum efficiency for fluorescence or phosphorescence is the ratio of the number of molecules that luminesce to the total number of excited molecule. Gives a measure of how efficient a fluorophore (i.e., fluorescing molecule) is. • A quantum yield = 1 means that every excited molecules deactivates by emitting a photon – such a molecule is considered a very good fluorophore. • Can express quantum yield as a function of rate constants Quantum Yield, = total # luminescing molecules total # of excited molecules k = rate constant]         k k k k k k k f f i ec ic pd d [ docsity.com Fluorescence and Structure • Low–energy   * (aromatic): most intense fluorescence. • Heterocycles do not fluoresce; heterocycles fused to other rings fluoresce. Heteroatom increases ISC then f decreases. • Conjugated double bond structures exhibit fluorescence. • Structural rigidity (e.g., naphthalene or fluorene vs biphenyl). Flexibility increases then f decreases. • Temperature: increase fluorescence intensity with decreasing T (reduce number of deactivating collisions). docsity.com (© 2007 Thomson Higher Education ds A “Viin cs C Hp» fluorene docsity.com • Solvent: increase fluorescence with increased viscosity (decreased likelihood of external conversion – radiationless deactivation) • Heavy atoms such as I, Br, Th increases ISC as a consequence f decreases • pH: Increased resonance structures (protonation or deprotonation)  stable excited state and greater quantum yield • pH can also influence emission wavelength (changes in acid dissociation constant with excitation) docsity.com H H H H H H H H | H \ Z \t/ \t7 Nv Ne N N Nt <> <> resonance forms of aniline anilinium ion © 2007 Thomson Higher Education ® docsity.com  EXCITATION AND EMISSION SPECTRA • Excitation spectrum: Emission wavelength is fixed; excitation wavelength is scanned – Monochromator or filters selected to allow only one  of fluorescent light to pass through to the detector. – Excitation wavelength is varied – at each excitation  increment fluorescent photons at the fixed emission  are collected. – The emission intensity (i.e., the number of fluorescent photons collected) at each  increment varies as the excitation  comes closer to or goes further from the  of maximum absorption  this is why an excitation spectrum looks like an absorption spectrum. docsity.com • Emission spectrum: Excitation wavelength is fixed; emission wavelength is scanned – Molochromator or filter is selected to allow only one  of excitation light to pass onto the sample. – Emission  is varied  fluorescent photons are collected at each incremental emission . – The emission intensity (i.e., the number of fluorescent photons collected) at each  increment varies as the emission  is changed. – Spectrum shows at what  the fluorescence intensity is a maximum for a given excitation . docsity.com Relative intensity Excitation Emission 200 250 300 350 400 450 500 550 600 Wavelength, nm docsity.com © 2007 Thomson Higher Education 1 Sample Electronics/ computer Emission Transducer A selector data system S| Excitation A selector Beam attenuator Teer Source © 2007 Thomson Higher Education docsity.com Lamp Ge !\ ! \\ { \\ Sample =} fF aperture Shutter E==5=5\\ disk 1} \\ A — Reference B Me photomultiplier Secondary Sample filter Fl] "Ore =a Seca | =i Mirror , 2 "Sample Reference aperture ._ disk n Higher Education docsity.com Emission monochromator Excitation monochromator Grating Reference photomultiplier tube aap Beamsplitter photomultiplier — white tube reflector Absorbance compensating cell Xenon lamp Sample (&) compartment 7 Thomson Higher Education docsity.com HO N A SY 8-hydroxyquinoline (reagent for Al, Be, and other metal ions) OH HO Sunt) . SO,Na alizarin garnet R ; (reagent for Al, F~) “h CO ~ ~OH Oo flavanol (reagent for Zr and Sn) 0 O Oto benzoin (reagent for B, Zn, Ge, and Si) ‘© 2007 Thomson Higher Education docsity.com TABLE 15-2 Selected Fluorometric Methods for Inorganic Species Wavelength, nm ——__—_—_——————_-___ LOD, Ton Reagent Absorption Fluorescence — pg/mL Interferences At Alizarin garnet R 470 500 0.007 Be, Co, Cr, Cu, F~, NO3, Ni, PO, >, Th, Zr F- Quenching of Al** 470 500 0.001 Be, Co, Cr, Cu, Fe, complex of alizarin Ni, PO, Th, Zr garnet R B,O;~ Benzoin 370 450 0.04 Be, Sb Caz 2-(o-Hydroxyphenyl)- 365 Blue 2 NH, benzoxazole Li? 8-Hydroxyquinoline 370 580 0.2 Mg Sn** Flavanol 400 470 0.1 Ba, PO,z, Zr Tet Benzoin - Green 10 B, Be, Sb, colored ions © 2007 Thomson Higher Education docsity.com