HANNAN ABDUL KAHLIQZMPI 16-04

PUNJAB UNIVERSITYZOOLOGY DEPARTMENT

SUBJECT: ADVANCED ANALYTICAL TECHNIQUES

Spectrofluorimetry“Determination of

Fluid volumes by Dye Dilution Technique”

Spectrofluorimetry

•Introduction– Principle– Purpose

•Phenomenon of fluorescence•Instrument•Factors affecting the rate of fluorescence

Introduction• Principle:– Some organic or inorganic compounds, when

hit by exciting radiation, emit different colored radiations, called fluorescence after de-excitation and can be detected. Emitted wavelength is always longer than incident one.

• Purpose:• Qualitative: Tells us what fluorescent species

is present using the wavelength of emitted radiation.

• Quantitative: Tells us how much of that species is present.

•Introduction– Principle– Purpose

•Phenomenon of fluorescence•Instrument•Factors affecting the rate of fluorescence

• Concept of Photoluminiscence:Emission of light after excitation.• Phosphorescence: Energy loss without light i.e.

heat• Fluorescence: Energy loss in the form of light

• Excited states of Electrons:– Singlet: Excited state of paired electrons, anti

parallel spin.– Triplet: Excited state of unpaired electrons,

parallel spin.

Phenomenon of Fluorescence

Single

t

Triplet

These excited states play a vital

role in fluorescence or

phosphorescence

10-710-4

•Phenomenon of fluorescence•Instrument•Factors affecting the rate of fluorescence

• Light source (Xe lamp striking UV light)• Monochromator (Desired wavelength)• Slit• Sample cuvette• Grating (selecting)• Photomultiplier• Detector• Computer

PARTS

Incident light

Absorption = Excitation

Stoke’s Law• “The wavelength of emitted light is always longer than the wavelength”

•Instrument•Factors affecting the rate of fluorescence

• Structure• Scatter• Quenchers• pH• Concentration• Intensity of incident light• Temperature and viscosity• Air (oxygen)

Factors

• Aromatic compounds are ideal• Compounds having π- π* transition fluoresce• Linear or highly conjugated molecules also

do but less than aromatic compounds• More the number of rings, more will be the

fluorescence• Fused ringed structure also fluoresce• Fusion of benzene rings to a heterocyclic

nucleus increases the absorption• Addition of halogens decreases the chances

of fluorescence because it increases the chance of phosphorescence

Structure

• Effect of structural rigidity:–More rigid the structure, more will be the

fluorescence.– Lack of rigidity will enhance the internal

conversion and hence “phosphorescence”.

Scatter• Rayleigh Scattering:– Re-Emission of small fraction of excitation light in

all directions form solute molecules.• Raman Diffusion:• Waves emitted by the solvent molecules as a result

of transfer of vibrational energy from solute molecules.

Incident Light Rayleigh Scattering

Raman Diffutsion

Quenchers• “Reduction of the fluorescence intensity

by the presence of substances in the sample other than the fluorescent analyte.”– Dynamic/Collisional quenching: direct Due to

collision– Static Quenching: Absorbance of

fluorescence

pH• Transfer of charge and hence the acidity

of a molecule affect the occurrence of fluorescence.

• More ionic a substance, more will be the fluorescence.

• For example: aniline has several resonance (ionic) forms but anilinium has only one.

Concentration• Quantum yield:

• For constant conditions, fluorescence is directly proportional to concentration.

(F) = (Io- IT) Ф• Value for highly fluorescenct substances

may be equal to one which is the Maximum value.

• If the concentration is too great, all light will be absorbed and the above expression will be:

F = IoФ

Where: Io = Incident light

IT = Transmitted light

Ф = Quantum yield of fluorescence

Intensity of incident light• Higher the intensity of light higher will

be the fluorescence.• If the intensity is too high too effects can

happen:– Spectral overlap– Photo decomposition: The molecule of

solution or changes its composition due to photochemical effect thereby affecting our results.

Temperature & Viscosity• Increase in temperature and decrease in

viscosity will affect the frequency of collision between the molecules and hence the fluorescence will be affected.

Air (Oxygen)• Direct oxidation of molecules can happen• Oxygen is a very effective quencher

Spectrofluorimetry“Determination of

Fluid volumes by Dye Dilution Technique”

• “Dye dilution or fluorescence derivatization is a technique in which some dyes (fluorimteric reagents) are added to such substance which cannot fluoresce and hence making to do so.”

• This technique is used to Qunatify some of the substances through fluorimetric techniques.

• fluorimteric reagents:– Organic aromatic reagents which are added

to non-fluorescence compounds for spectrofluorimetry.

Examples

• Determination of primary amines, amino acids, peptides etc. through reaction with fluorescamine reagent:

Fluorescamine Amino Acid

Fluorescent compound

Determination of primary and secondary aliphatic amines through:

a) Reaction with 4-chloro-7-nitrobenzo-2-oxa-l,3-diazole ( NBD-CI ) give yellow fluorescence:

b) Reaction with l-dimethylaminonaphthalene-5-sulphonyl chloride (Dansyl, chloride):

• Thiamine HCI in pharmaceutical preparations such as tablets and elixirs and in food stuffs such as flour is relatively easily determined by oxidation to highly fluorescent thiochrome. The product is soluble in 2-methyl-propan-1-ol and hence is easily extracted from the reaction mixture for measurements.

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