Chapter 15

Molecular Luminescence Spectrometry

A very sensitive and selective instrumental technique with some of the lowest LOD's for molecules that luminesce (not all do!) - down to single-molecule detection.

A very sensitive and selective instrumental technique with some of the lowest LOD's for molecules that luminesce (not all do!) - down to single-molecule detection.

diamagnetic diamagnetic paramagnetic

isoelectronic in external magnetic field

isoelectronic splits into 3 energy levels

short (ns) lifetimes;

"allowed"longer (s or longer) lifetimes; "forbidden"

large small

Note: fluorescence, phosphorescence, and non-radiative decay can all occur simultaneously depending on the "quantum yields".

Note: fluorescence, phosphorescence, and non-radiative decay can all occur simultaneously depending on the "quantum yields".

ps ps

(Jablonski Diagrams)

The quantum yield for a given process is the ratio of the number of molecules that undergo a particular process to the total number of excited states.

The quantum yield for a given process is the ratio of the number of molecules that undergo a particular process to the total number of excited states.

states excited total

fluoresce molecules numberf

states excited total

cephosphores molecules numberp

1.00i

So

S1

T1

Kasha's Rule: fluorescence in fluid solution always occurs from the lowest vibrational level of the 1st excited singlet state.

Kasha's Rule: fluorescence in fluid solution always occurs from the lowest vibrational level of the 1st excited singlet state.

So ()

collisions

C = OH

H

.

:* e-eC O

H

H

::

e-vC = O

H

H

::

~~

solvent molecules

S1 (*)

So ()

S1 (*)

e-t

So

S3

S2S1

Favored by overlapping vibrational levels.

So

S1

T1

Change in spin state from a singlet to a triplet and vica-versa.

Heavy atom effect – atoms and small molecules such as Hg, Br2, I2, and O2 have large “spin-orbit coupling” that helps flip spin.

Heavy atom effect – atoms and small molecules such as Hg, Br2, I2, and O2 have large “spin-orbit coupling” that helps flip spin.

k

1 lifetime eCC kt

o

exci

ted

stat

e po

pula

tion

C →

time →

Co

oo

oo 0.37Ce

C

k

1k- expC k- expCC

o0.37C

•unique and useful for identification

•inversely proportional to

•Co = initial population of the excited state

•k = 1st order rate constant (units s-1)

Most intense from molecules containing multiple aromatic rings, →* transitions, and structural rigidity.

Most intense from molecules containing multiple aromatic rings, →* transitions, and structural rigidity.

no fluorescence

fluorescence

f = 1.0

f = 0.2

excitation spectra – measure luminescence at a fixed wavelength while scanning the excitation wavelengths.

emission spectra - excite at a fixed wavelength while scanning the luminescence.

excitation spectra – measure luminescence at a fixed wavelength while scanning the excitation wavelengths.

emission spectra - excite at a fixed wavelength while scanning the luminescence.

overlap at the 0-0 band.

http://en.wikipedia.org/wiki/Image:Xenon_short_arc_1.jpg

The absorbance compensating cell contains the dye molecule Rhodamine B which absorbs all wavelengths equally and emits in a pattern that mimics the variation in the lamp output. The corrected excitation spectra are produced by dividing the raw spectrum with the Rhodamine B spectrum.

The ability to distinguish and identify different molecules based on differences in luminescence spectra. Frequently enhanced by cooling the sample.

The ability to distinguish and identify different molecules based on differences in luminescence spectra. Frequently enhanced by cooling the sample.

Po Pt

Pf

a

ff

P

P

states excited total

fluoresce molecules number

Pf = fPa = f(Po – Pt) = fPo(1 – Pt/Po)

from Beer’s Law:Pt

Pa

= 10-bc

so Pf = fPo(1 – 10-bc)

Converting to base e and expanding e-bc in a MacLaurin Series* –

Pf = fPo(1 – 2.303e-bc)

Pf = fPo[2.303bc – (2.303bc)2/2! +

(2.303bc)3/3! +…..

Pf = fPo 2.303bc

*

Pf = fPo 2.303bcA = -log P/Po

Why fluorescence is inherently more sensitive.Why fluorescence is inherently more sensitive.

anthracene (MW = 178)

1. Fluorimetric Determination of Inorganic Species

2. Fluorimetric Determination of Organic Species

3. Forensics (latent blood stains - luminol chemiluminescence)

4. Fluorescence Imaging Methods

Polynuclear Aromatic Hydrocarbons (PAH's) in the environment.Polynuclear Aromatic Hydrocarbons (PAH's) in the environment.

perylene

1,2-benzanthracene

anthracene fluorescence LOD phosphorescence LOD (parts per trillion) (parts per billion)

3 3

8 0.1

1 ---

http://en.wikipedia.org/wiki/Luminol

http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html