Fluorescence spectrscopy

• Analytical technique used to investigate/measure the light energy

• FLUORESCENT COMPOUND = Compound that exhibits FLUORESCENCE i.e. Compound that can emit light (photons) following the absorption of EM radiation

• Fluorescence requires the initial excitation of the compound molecules using an

• Molecules absorb radiation then after a very short time period will emit radiation of lower energy and of longer λ

• involves molecular excitation from a ground state energy level to a higher level i.e. absorption of radiation

• Excited molecules will lose energy (mostly via molecular collisions) and hence begin a gradual ‘step-wise’ transitional decay (descent back) to their initial ground state level

• Latter decay process is ultimately accompanied by emission of low energy/high λ radiation mostly as visible radiation

• Emitted radiation is of lower energy than the energy initially absorbed - a phenomenon known as the STOKES SHIFT

• energy level diagram for fluorescence - absorbance of light to go into the excited states and radiationless transition falls back to ground states, emission of light

basics

• Typical energy of excitation applied to give the strongest/best fluorescence roughly corresponds to the λmax of absorption

• emitted radiation will usually be shifted by λ of 50-150 nm

• emission/Fluorescence spectrum of a compound represents a plot of all the energy emissions λ and approximates a ‘mirror image’ of the compound UV-visible absorption spectrum

measuring energy excitation

• measures the efficiency of a compound's fluorescence—essentially acting as the "percentage yield" of emitted light versus absorbed light.

• number of photons emitted/number of photons absorbed

• For majority of fluorescent compounds φ = 0.3-0.7

• Strongly fluorescent compounds possess φ close to 1 and non-fluorescent compounds possess φ =0

• Various types of compounds display fluorescent behaviour

Fluorescence Quantum Yield

• Predicting fluorescent activity based on chemical structure is problematic - generally most fluorescent compounds possess

• EXTENDED CHROMOPHORE &/OR AUXOCHROME SYSTEM

• RIGID &/OR PLANAR CHEMICAL STRUCTURE

• features constitute the compound FLUOROPHORE and typically associated with aromatic/polycyclic compounds

• Many phenolic compounds exhibit good fluorescence - estrogen

• Various types of fluorescent drugs exist e.g. catecholamines, steroids, fluoroquinolones, antimalarial quinolines, profens, tricyclic antidepressants, antihypertensives, SSRIs, vitamins

• several non-fluorescent drugs can be transformed into fluorescent derivatives via a chemical derivatisation process

• Generally involves the addition of a fluorescent group using a fluorescent derivatisation reagent

• less drugs have fluorophores compared to chromophores

• limited technique and less ability to be applied for drugs than uv spectrophotometry

predicting fluroescent activity

• fluorimetric assays are very sensitive and selective

• used for QUANTITATIVE and qualitative analysis

• quantitative assays require the use of very dilute sample and ‘external standard’ solutions (comparative assays)

• various problematic factors to consider - linked mostly to analyte concentration, solvent and light scattering effects

• dependent on sample type may require sample clean-up or a separative process prior to analysis (e.g. LLE, SPE, HPLC)

• Instrument used for analysis is the fluorimeter (or fluorescence spectrophotometer) - similar in design to UV spectrophotometer

• temperature and pH dependent

• solvent scattering the light - raman or rayleigh scatter

• particles scattering light -Tyndall scatter

• halide ions can absorb the fluorescence coming out of your sample

fluorescence assays

• Generally employed to specifically measure very low drug concentrations in a wide variety of biomedia and formulations

• used to assess drug impurities and drug metabolites

• Broad range of pharmaceutical applications include:

• drug formulation analysis

• drug impurity limit testing, drug stability and dissolution studies, drug bioanalysis, drug binding studies

• Widely used technique in clinical/biomedical analysis

• Fluorimeters are also often utilised as drug detectors within chromatographic instruments (mostly for HPLC)

applications