INTRODUCTION TO SPECTRAL METHODS OF ANALYSIS

Maam Glenn Medina De La Salle University

What happens when a molecule absorbs light?

2

So = Ground state S1 = Excited state (Singlet) T1 = Excited state (Triplet)

3

Vibrational Relaxation (R1)

Radiationless transition
Energy is transferred to other molecules through collision Vibrational energy converted to heat

Internal Conversion (IC)

Radiationless transition between states with the same spin quantum numbers
S1 So

Vibrational Relaxation (R2)

Radiationless transition (within So)
Energy is transferred to other molecules through collision

Intersystem Crossing (ISC)

Radiationless transition between states with different quantum numbers
S1 T1

Vibrational Relaxation (R3)

Radiationless transition (within T1)
Energy is transferred to other molecules through collision

10

Intersystem Crossing (ISC)

Radiationless transition between states with different quantum numbers
T1 S0

11

Vibrational Relaxation (R4)

Radiationless transition (within So)
Energy is transferred to other molecules through collision

12

Fluorescence
Emission of photon during transition between states with the same spin quantum numbers (S1 S0)

13

Phosphorescence
Emission of photon during transition between states with different spin quantum numbers (T1 S0)

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Factors Affecting Transitions

1. Nature of substance
2. Nature of solvent 3. Temperature 4. Pressure

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Fluorescence All Around Us

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Fluorescence vs Phosphorescence
1. Fl shorter lifetime
2. Ph rarer than Fl 3. Ph longer wavelength 4. Fl 10x stronger than Ph

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LUMINESCENCE
1. Emission of light from an excited state of molecules 2. Examples: Fl and Ph 3. More sensitive than absorption more sensitive enough to observe single molecules

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Absorption and Emission Spectra

Eabsorbed > Eemitted

Wavelengthabsorbed < Wavelengthemitted

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Excitation and Emission Spectra

Emission Spectrum Emission intensity vs Emission wavelength (irradiance measured) Excitation Spectrum Emission intensity vs Excitation wavelength

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Excitation and Emission Spectra

The sample is irradiated at 1 l and emission is observed over a range of ls. Excitation monochromator selects excitation l Emission monochromator selects 1 l at a time

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Fluorometric assay of Se in Brazil Nuts

Luminescence in Analytical Chemistry

Fluorescein a strongly fluorescent compound that can be coupled to many molecules for analytical purposes

Luminescence in Analytical Chemistry

Forensics fingerprint labeling Calcium detection

DNA microarrays mutations, pathology

DNA strand tagged with fluorescent labels

Chemiluminescence
Emission of light arising from a chemical reaction Fireflies Light stick Jellyfish Nitric oxide determination (luminol) Calcium analysis Thyroxine determination

End of Lecture 3!
Answer:

18-D
18-1

18-2
18-12

18-18