Fluorescence Spectroscopy

Question 1

What is Fluoroscence Spectroscopy?

Answer 1

Analytical technique used to investigate/measure the light energy emitted from a fluorescent compound

Question 2

What is a fluorescent compound?

Answer 2

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

Question 3

Explain how flurosence works

Answer 3

• Fluorescence requires the initial excitation of the compound molecules using an external source of UV/visible radiation
• Molecules absorb radiation then after a very short time period will emit radiation of lower energy and of longer λ
• Initially 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

Question 4

What is the wavelength required to give strongest/best fluoroscene

Answer 4

λ_{max of absorption} of absorption

• Emitted radiation will usually be shifted λ of 50-150 nm
• Emission/fluorsence spectrum of a compound represents a plot of all energy emissions λ and approximates a mirror image of the compoud UV-visible and absorption spectrum

Question 5

How is fluroscence efficiency measured of a compound/drug?

Answer 5

Measured by fluorescence quantum yield

ϕ =NO. OF PHOTONS EMITTED / NO. OF PHOTONS ABSORBED

• majority of fluorescent compounds ϕ = 0.3-0.7
• Strongly fluorescent compounds possess ϕ close to 1
• non-fluorescent compounds possess ϕ= 0
• Various type of compounds display fluorescent behaviour

Question 6

Predicting fluorescent activity based on chemical structure is problematic, what structures do most fluroescent compound possess? What compound does this form

Answer 6

• EXTENDED CHROMOPHORE AND/ OR AUXOCHROME SYSTEM
• RIGID AND/OR PLANAR CHEMICAL STRUCTURE

Above features constitute the compound fluorophore and typically associated with aromatic/polycyclic compounds

> Many phenolic compounds exhibit good fluorescence

Question 7

A. What are some fluorescent drugs that exist?

B. How can non-fluoroscent drugs be converted to fluorescent derivatives

Answer 7

A)

• catecholamines, steroids, fluoroquinolones, antimalarial quinolines, profens, tricyclic antidepressants, antihypertensives, SSRIs, vitamins

B)

• Via a chemial derivatisation process
• involves the addition/transfer of a fluorescent group using a fluorescent derivatisation reagent (e.g. dansyl chloride)

Question 8

For assays and instrumentation of FS;

A) Is it used for quantitative or qualititative analysis

B) What is the equation used to quantify drug amount

C) What are some problem factors

D) what is the instrument used

Answer 8

A)

Both

B)

F = I_o x 2.303 x A1^%_1cm x c (in g/100 mL) x l _{(in cm)}

F: fluroscence emitted

I_o: Intensity of incident light

C)

• 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)

D)

• Fluorimeter (or fluoroscence spectrophotometer) –> similar in design to UV spectrophotometer

Question 9

What are some pharmaceutical applications of fluroscence spectroscopy?

Answer 9

• specifically measure very low drug concentrations in a wide variety of biomedia and formulations
• assess drug impurities and drug metabolites
• 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)