LUMINESCENCE

Definition : Light other than black-body radiation emitted by a sample.

Photoluminescence - excitation by an external light source.

Bioluminescence - e.g. firefly

luciferase
ATP + lucifern + O2 ⎯⎯⎯→ AMP + PP + oxylioferin + CO2 + H2O + hν
Mg2+

Chemiluminscence -

Advantages of Luminescent Methods

• Versatility

Sample types : organic, inorganic, synthetic, natural, small and large

molecules.

Sample formats : solutions (concentrated or dilute), gases, suspensions, solid

surfaces.

• Combination with other methods : hplc. tlc, microscopy.

1. Sensitivity

In solution , pg ml-1 (cf. µg ml-1 for AAS)

2. Selectivity

Fluorescence : Absorption & emission wavelength to characterise a sample.

Phosphorescence : also lifetime and P:F ratios.

Combined methods (Biochemical Analysis)

(i) “spectroscopic tricks” - derivative spectroscopy

(ii) combination with separation methods
(iii) combination with biological reactions, e.g. immunoassay, enzyme assay.
Energy States

S1

T1

S0

S1 - excited singlet
T1 - excited triplet
S0 - ground state singlet.

Deactivation of an Excited Molecule

(non-luminescent)

1. Internal Conversion

Excess energy is converted to vibrational energy, which is lost as heat.

2. Collisional Quenching

S1 + Q ⎯→ complex ∼∼∼∼→ S0 + Q + heat

The Process of Fluorescence

1. Absorption of photon - gain in electronic and vibrational energy.

S0 ⎯→ S1 10-5 secs

2. Internal conversion and vibrational relaxation to the lowest vibration level of S1.

10-12 secs (may remain for ca. 10ns)

3. Return to ground state by radiative transition.

fluorescence (10-9 - 10-10 secs)

Fluorescence has a larger wavelength (lower energy) than absorption - “STOKES

SHIFT”.

The Process of Phosphorescence

1. Absorption S0 → S1

2. Conversion to lowest vibrational level of S1

3. “Intersystem crossing” to the lowest triplet state T1 (lower energy than S1)

4. Return to ground state by radiative transition.

Phosphorescence (10-3 - 10-2 secs). Long lifetime due to forbidden transition

S1 → T1

(a) Observed at 77K

(b) at room temperature - solid surface or micelle (protect from collisional
quenching).

Other Mechanisms for Loss of Energy

1. Photodecomposition
2. Energy transfer (especially biological systems)

Fluorescence and Structure

Fluorescent compounds contain :-

(i) aromatic functional groups (low energy π → π* transitions)

(ii) aromatic or aliphatic carbonyls
(iii) highly conjugated double bond systems

Structure Effects with Aromatic Compounds

(i) fluorescence increases with the number of rings.

(ii) simple heterocyclics - non-fluorescent
fused ring structures - fluorescent
(iii) substitution : (a) fluorescence decreases with increasing halogen size
(b) carboxylic acids reduce fluorescence

Structural Rigidity

Qf = quantum efficiency

Inorganic Compounds
Unchelated luminescent ions : uranyl ion UO2+
thallium(I) ion.

Aquated Tl+ , excitation 215nm, weak emission 370nm.

Add 1M KCl ⇒ TlCl42- ion, excitation 240nm, strong emission 430nm

Chelates of non-Transition Metal Ions

e.g. 8-hydroxyquinoline

Groups IA IIA IIB IIA IIIB and Zr4+ determined as chlorides.

Most frequently IIIA, aluminium and gallium.

Instrumentation

Very important to have 90° optics so that light doesn’t shine directly on detector.

Sources

Properties required :

a. intensity
b. wavelength distribution
c. stability

1. High pressure dc xenon arc lamp

continuum 300 → 1300nm (several strong lines 800 → 1100nm)

arc compressed between electrodes.

2. Xenon flash lamp

high energy flash produced by the discharge of a capacitor through a lamp

filled with xenon.

Cells
1cm square, 4.5cm high. Synthetic fused silica (low natural fluorescence → 190nm).
Plastic polystyrene. Glass → 320nm.

Matt black cell compartments. Also flow cells available.

For bioluminescence and chemiluminescence, there is no need for a light source or

excitation monochromator.

PHOSPHORESCENCE

Longer lived emission that may occur alongside fluorescence.

To measure : 1. Pulse with light

2. Cut off source
3. Measure emission

Experimental Conditions

Liquid nitrogen temperature , 77K. Solvent EPA (mixed solvent) :

Diethyl ether : Isopentane : Ethanol

5 5 2

This gives a transparent glass at 77K.

Apparatus :

Procedure : Make solution in water, methanol, cyclohexane, such that UV/vis at

max ≈ 0.04.
Set excitation monochromator to the maximum absorbance wavelength.
 Set emission monochromator to the maximum absorbance wavelength +
10nm.
Measure emission to 650nm.

If fluorescent, will see similar to a normal UV/vis peak.

Check for true excitation maximum :-

Set emission monochromator to the maximum.

Set excitation monochromator to UV/vis max - 20nm.
Scan excitation monochromator for 60nm.

Scattering

Rayleigh - by the molecules themselves.

Tyndell - by small particles.

Rayleigh

Occurs at excitation wavelength and at multiples of that wavelength.

Raman

Conversion of energy to vibrational and rotational energy to a longer wavelength.

Raman, since the energy abstracted for a given Raman band is always constant, the
separation from excitation wavelength is constant, and irrespective of wavelength.

Factors Affecting Fluorescence Intensity

1. pH
2. Temperature
3. Viscosity
4. Solvent
5. Quenching

pH Effect

Often only one ionic form of a molecule is fluorescent.

Protonation reactions have rate constants higher than fluorescence. Hence, many
absorb as a neutral molecule and fluoresce as an ionised molecule.

Temperature Effect
Increasing temperature reduces fluorescence because of an increase in collisional
quenching. This can be 5% per °C, so thermostat cells should be used for sensitive
measurements.

Viscosity

Increasing viscosity increases the fluorescence, because collisional quenching is

reduced. This may be deliberately used in fluorescence polarisation.

Effect of Solvent

Large and unpredictable effects on intensity and wavelength.

Quenching

Molecular interactions reducing fluorescence quantum yields. Quencher, Q, may form

complexes with ground state molecules (static quenching) or with excited state
(dynamic quenching).

Sterm-Volmar equation :

For static quenching, K is related to the equilibrium constant of the complex.

For dynamic quenching, K is the product of the rate constant of quenching and the
fluorescence lifetime.

Fluorescence Intensity

Fluorescence is measured against a black background ⇒ sensitivity.

For a dilute solution, from the Beer-Lambert law.

Amount of light absorbed =

I0 = incident radiation
ε = molar absoptivity
b = path length
c = molar concentration

Quantum yield,
 if absorbance is small

So, fluorescence is proportional to I0 (light source) and to concentration.

In practice, holds for limiting circumstances : absorbance = 0.05, deviation of 5%
from linearity.

FLUORESCENT LABELS

Compound X + Fl-Label ⎯⎯⎯⎯→ X-Fl

(non-fluorescent) (may fluoresce) (fluorescent)

Fluorescamine

4-phenylspiro[furan-2(3H),1’-phthalan]-3,3’-dione

1 Fluorescent & 1 Non-Fluorescent

e.g. riboflavin and thiamine (in vitamin pill)

Riboflavin
ex. 435nm em. 530nm
Measure fluorescence directly.

Thiamine
Oxidise to thiochrome and measure at ex. 366nm and em. 460nm.

Organic Derivatisation

REAGENT REACT WITH λex λem

Dansyl-Cl 1°, 2° amino, phenolic OH 298 545
Bansyl-Cl Aminos 300 530
OPT Peptides, 1° aminos 350 470
Fluoram 1° aminos 390 450
NDB-Cl 1°, 2° aminos 480 550
BR-Mac carboxylic acids 328 380
EDTN aliphatic OH 350 420

Chromatographic Detection

TLC - Pre-coated sheets with inorganic phosphors - luminescence is quenched by

organic compounds ⇒ dark spots (inner filter effect). Green phosphors (522nm) : (i)
zinc silicate (ii) cadmium silicate with Mn/Pb activator. Excitation 254nm, longer
wavelength used for fluorescent organics.

Quantitative methods by measuring luminescence or quenching.

Advantages of Fluorescence Detection

1. Sensitivity a. highly fluorescent label

b. pre-concentration prior to injection

2. Selectivity

Pre-Column Derivatisation
Advantages : Solvent system unrestricted
Reaction conditions unrestricted
Can be used as a clean-up step
Wide choice of reagents

Disadvantages : Possible formation of artefacts or several derivatives of a

compound
Individual preparation
Internal standard required
Reproducibility may be low because of inconsistent reaction

Post-Column Derivatisation

Advantages : Sample preparation minimal

Derivatisation automated, precision high
Formation of artefacts much less likely

Disadvantages : Restriction on eluent (insolubility or reactions)

Reaction must be rapid (<30 seconds)
Resolution may be adversely affected by post-column mixing

Dansyl Chloride

Primary and secondary amino groups and phenolic -OH.

Reaction - slow at room temperature.

Use - pre-column derivatisation.

Properties - λex 350nm

λem 450-580nm (longer wavelengths in polar solvents)

Experimental - 2µl amino acid solution

20µl pH 10.5 buffer
50µl DNS-Cl

Heat at 100°C for 2 minutes

Inject 10µl

Fluram, Fluorescamine

Primary amino grounds (non-fluorescent label, hydrolysed to non-fluorescent

product).
Reaction - (half time) 100-500ms

Use - post-column derivatisation.

Properties - λex 390nm

λem 490nm

Experimental - 0.03% w/v solution in water-free acetone, effluent adjusted to pH 8.5 -

9.0 (borate buffer 0.01M), reagent added with second pump.

Detection limits : 5-10mg peptides per injection

PHOSPHORESCENCE

Work at 77K
Room temperature - reduces luminescent molecules.

1. Simple chemical additions to the solvent.

Sometimes make these additions at 77K.

- promote heavy atom effect.

e.g. by adding Br, I to solution.

Fluorescence/phosphorescence ratio pushed towards phosphorescence. Promotes
inter-system crossing.

e.g. F : P

10 : 1
with heavy 1 : 1
atom

- add sodium sulfite. Stops oxygen quenching of phosphorescence.

Usually add at 77K, but 4 or 5 examples where adding both may give
phosphorescence at room temperature.

2. Forming inclusion complexes

(a) Form a micelle in solution so that the actual phosphorescent molecule sits inside
the micelle. Micelle protects molecule from collisional quenching. Can sometimes see
phosphorescence at room temperature.

(b) Cyclodextrins. “Bucket shaped molecule”, has hydrophobic interior. Available as

α, β, γ cyclodextrins. Only main difference in the size of cavity. Phosphorescent
molecule sits inside the “bucket” and is protected from collisions.
(c) Solid supports. Immobilise phosphorescent molecule on a solid surface, e.g. dip
filter paper into phosphorescent solution, dry, and measure phosphorescence. Latex,
plastic tubes. Collisional prevention.

3. Work on solid samples.

Applications of Phosphorescence

Immunoassay

Examples using iclusion complexes and solid supports.

Advantage of no background over fluorescence.

Clinical Chemistry

Serum has fluorescence spectrum itself, even at 1:200 dilution, get considerable
fluorescence in UV/vis region. Very short lived.

Environmental Chemistry

Very complex mixture. May have aromatic components - many of these fluoresce.

Irradiate sample, allow fluorescence to decay - measure phosphorescence.

Background from serum etc. decays away.
Very rare to get phosphorescence in environmental samples, none from serum.

Automatic Sorting in Post Office

Stamp is phosphorescent to orientate letter.

CHEMILUMINESCENCE

Measure against totally black background ⇒ sensitivity.

Applications

Enzyme Immunoassay

cortisolhorseradish peroxidase luminol / H2O2

thyroxin glucose oxidase glucose / TCPO-ANS
insulin glucose oxidase lactose / TCPO-ANS
Detection limit 1x10-15 mol. Much better than fluorescence immunoassay due to no
background.

Lucinigen shows chemiluminescence on oxidation. This occurs via the reducing

sugars.
Don’t lose any light through optics, monochromators etc. Only consists of a cell and a
detector.

Chemiluminescence Energy Transfer

Measure fluorescence. Can use dedicated clinical analysers that work at one fixed
wavelength. May give better quantum yields.

Flow-Injection

• Must have rapid mixing prior to assay.

• Can use reagent stream to regenerate enzyme.
• Rapid and reproducible mixing improves sensitivity.
• Rapid sample throughput

Continuous Flow Analysis (Autoanalyser)

Enzymes are immobilised on nylon.

E1 - conversion of analytes to product 1

E2 - conversion of product 1 to product 2, producing chemiluminescence.

HPLC

Quenching of chemiluminescence by sulfites, anilines, organosulfur compounds.

ng levels. Linear range 2-3 orders of magnitude.

When only HPLC solvent coming off column, get luminescence.

When quencher comes off, get negative peak.