Spectra - Advanced Analytical Chemistry - Lecture Slides, Slides of Analytical Chemistry

Download Spectra - Advanced Analytical Chemistry - Lecture Slides and more Slides Analytical Chemistry in PDF only on Docsity! Spectra 1) Excitation Spectrum – fluorescence or phosphorescence intensity (at fixed λ) as a function of excitation λ or absorption λ 2) Fluorescence Emission Spectrum - fluorescence emission intensity vs. λ for a fixed excitation λ (= absorption λ, max.) 3) Phosphorescence Emission Spectrum – phosphorescence emission intensity vs. λ for a fixed excitation λ (= absorption λ, max.) docsity.com Phenanthrene Spectra Relative intensity Fluorescence Ll Excitation 200 300 400 500 600 Wavelength, nm ® docsity.com  EEMs represented as contour plots | 1,00 M KCl FIGURE 3. Absorbance-corrected EEMs of PFAR (50 mg/L, pH 6.0) ( different ionic strengths. 3 docsity.com Spectra 5) Synchronous Scan Spectrum – record fluorescence intensity while scanning both excitation and emission λ, usually at a constant offset (∆λ) Synchronous scans of natural organic matter with ∆λ = 25 nm at pH 4 & 9 (solid line) docsity.com Fluorescence Efficiency or Quantum Yield (ФF) Number of photons emitted ФF = -------------------------------------------Number of photons absorbed Fluorescence efficiency depends on the rate of fluorescence relative to the rates of other processes involving excited state deactivation KF 1ФF = ----------------------- KF = ---- KF + KIC + KIS τF rate of fluorescence rate of radiationless deactivation or internal conversion rate of intersystem crossing fluorescence lifetime docsity.com Inner Filter Effects – if the absorbance of the sample is above 0.05, fluorescence intensity is decreased in two ways: 1) Primary Inner Filter Effect – excitation energy is decreased by absorption at the λ of excitation which decreases fluorescence 2) Secondary Inner Filter Effects – emitted fluorescence is absorbed by the sample at the emission λ which also reduces the amount of fluorescence measured docsity.com Primary Inner Filter Effect At high ε b C The front part of the cell is illuminated more efficiently than the back part
get less fluorescence from the back part of the cell Incident Beam Io Sample Cuvette Transmitted Beam I Represents Absorption i.e. inner filter effect docsity.com Inner Filter Effects – conclusions - Inner filtering results in curved calibration curves - Must know or measure absorbance at both the excitation and emission wavelengths - Fluorescence is basically a trace technique having 1 - 3 orders of magnitude lower detection limits than absorbance - Upper limit of fluorescence occurs around lower limit of absorbance (complimentary) - Inner filtering is also called quenching docsity.com Effects of other solutes on excited state emission
can promote radiationless return to ground state = quenching 1) Energy Transfer 1A* + Q
A + 1Q* or 1A* + Q
1{AQ}* Occurs by a) Q absorbs the emission of A b) Collisional non-radiative transfer of energy
obeys Stern-Volmer Law Where A = Analyte (species of interest) which absorbs and Q = quencher 1A* & 1Q* designate first excited singlet & 1{AQ}* = complex docsity.com Stern-Volmer Equation ΦFo ------ = 1 + KQ τFo [Q]ΦF Two types of quenching are covered by the Stern-Volmer Equation: 1) Static quenching – where A & Q form a complex that doesn’t emit (=dark complex) 2) Dynamic quenching – collisional Both represented as 1A* + Q
1{AQ}* (increased rate of radiationless deactivation KIC) Fluorescence Quantum Yield in the absence of quencher With quencher Quenching constant Fluorescence Lifetime of A (no Q) Concentration of Quencher docsity.com Fluorescence Quenching of Fulvic Acid, Q = Cu2+ Hays, 1996 Emission scans of 15 mg/L Fulvic Acid with varying [Cu2+] and excitation at 340 nm docsity.com Equations for Fitting Data Equation for One Site Binding T=[200+2K TpyssCyrTnssl(KC,t+KCyt1)-(KCtK Cyt 14K? Cy CT] M[2+2K Cy [OKC AK Cyt 1)-(KC,+K Cyt 14K, Cu) T] Equations for Two Site Binding Cua = [M] + (K,Cy [M/K,[M] + 1)+(K,C,,[M)/K,[M] + 1)+.... +K,C.n[M)/K,[M] + 1 KK, [MP + {K,K,(C,,+C,y-Cy) + K, + K} [MF + {C.K +K,C,,-Cy(K, + K,+1}[M] - Cy=0 docsity.com Fluorescence Quenching Curves Hays, 1996 15 mg/L of Fulvic Acid with varying Cu(II) concentrations at pH 5 (o), 6 (∇) & 7 (
 Added Copper Concentration (M x105) R el at iv e Fl uo re sc en ce In te ns ity docsity.com Fluorescence binding curves for Cu, Co, Mn & Al at pH values of 5-8 docsity.com Excitation Emission Matrix 15 mg/L Fulvic Acid at pH 4 with no Al(III) docsity.com Excitation Emission Matrix 15 mg/L Fulvic Acid at pH 4 with Al(III) docsity.com Fluorescence Enhancement Curve with One-Site Model 15 mg/L of Fulvic Acid with increasing Al(III) concentrations at pH 4 (o) docsity.com