April01_2005_2pp

April01_2005_2pp - • Emission • modest resolution •...

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1 April 1, 2005 ¾ April Fool’s Day Cancelled Due to Leap Year / 2 Effect of Concentration I f = Φ (I o –I) Fluorescence Intensity Fluorescence Quantum Efficiency Incident Radiation Intensity Transmitted Radiation Intensity
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3 Substitute and Simplify Beer’s Law: I=I o e - ε bc Gives: I f = Φ I o (1-e - ε bc ) Expand: I f = Φ I o ε bc [1-( ε bc/2!)+(( ε bc) 2 /3!) . . . ] If ε bc is small (<0.05) , then: I f = Φ I o ε bc 4 Analytical Implications I f = Φ I o ε bc ¾ At specific λ exc with constant I o : I f =Kc ¾ I f I o So, signal will increase with increased source intensity
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5 Quantitative Considerations Concentraton I f small ε bc medium ε bc large ε bc (I f ≈Φ I o ) actual 6 Instrumentation Source Excitation Dispersive Device Sample Emission Dispersive Device Detector I o λ ex I f λ em
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7 Sources Continuum ¾ Xe Arc lamp • Intense UV/Vis source Line ¾ Hg Vapor Lamp ¾ Laser •N 2 - or Nd:YAG-pumped tunable dye laser • Ar+ or Kr+ laser 8 Dispersive Devices ¾ Excitation • Only needed for continuum sources • modest resolution, high light throughput
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Unformatted text preview: • Emission • modest resolution • high stray light rejection (double-monochromator) 9 Typical Spectrofluorometer 10 Applications ¾ Quantitative Analysis • Need molecule with high fluorescence quantum efficiency OR one that can react quantitatively to form a highly fluorescent compound • Linear relationship with concentration only at low concentrations • Fluorescence signal directly proportional to source intensity: Laser sources should give best results 11 Laser-Excited Fluorescence Why use Laser Sources? ¾ Saturation Effects • I f indep of I o – variation of source intensity not observed in signal – decreased noise • is maximized – increased signal – best detection limits (ppt and lower) • Extends LDR – by lowering detection limits...
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This note was uploaded on 05/15/2010 for the course CHEM 221 taught by Professor Joelgoldberg during the Spring '10 term at UVM.

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April01_2005_2pp - • Emission • modest resolution •...

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