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

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

T1

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 ⇒ TlCl4

2- 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 cortisol horseradish 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.