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INTRODUCTION TO

SPECTRAL METHODS OF ANALYSIS

Ma’am Glenn Medina

De La Salle University

What happens when a

molecule absorbs light?

S o = Ground state

S 1 = Excited state (Singlet) T 1 = Excited state (Triplet)

Vibrational Relaxation (R 1 )

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 S 1 S o

Vibrational Relaxation (R 2 )

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

Intersystem Crossing (ISC)

Radiationless transition between states with different quantum numbers S 1 T 1

Vibrational Relaxation (R 3 )

Radiationless transition (within T 1 ) Energy is transferred to other molecules through collision

Intersystem Crossing (ISC)

Radiationless transition between states with different quantum numbers T 1 S 0

Vibrational Relaxation (R 4 )

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

Fluorescence

Emission of photon during transition between states with the same spin quantum numbers (S 1 S 0 )

Phosphorescence

Emission of photon during transition between states with different spin quantum numbers (T 1 S 0 )

Factors Affecting

Transitions

1. Nature of substance

2. Nature of solvent

3. Temperature 4. Pressure

Fluorescence All Around Us

Fluorescence vs Phosphorescence
Fluorescence vs
Phosphorescence

1. Fl shorter lifetime

  • 2. Ph rarer than Fl

  • 3. Ph longer wavelength

  • 4. Fl 10x stronger than Ph

LUMINESCENCE
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

Absorption and Emission

Spectra
Spectra

E absorbed > E emitted

Wavelength absorbed < Wavelength emitted

Excitation and Emission Spectra
Excitation and Emission Spectra

Emission Spectrum Emission intensity vs Emission wavelength (irradiance

measured)

Excitation Spectrum Emission intensity vs Excitation wavelength

Excitation and Emission Spectra
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

Fluorometric assay of Se in Brazil Nuts
Fluorometric assay of Se in
Brazil Nuts
Fluorometric assay of Se in Brazil Nuts
Luminescence in Analytical Chemistry
Luminescence in Analytical
Chemistry

Fluorescein a strongly fluorescent compound that can be coupled to many

molecules for analytical purposes

Luminescence in Analytical Chemistry
Luminescence in Analytical
Chemistry

Forensics fingerprint labeling

Calcium detection

DNA microarrays mutations, pathology

DNA strand tagged with fluorescent

labels

Chemiluminescence
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!
End of Lecture 3!

Answer:

18-D

18-1

18-2

18-12

18-18