fluorimetry basic idea

BYSUBHASISH DAS

M.PHARM 1ST YR.

Luminescence is the term applied to the remission of previously absorbed radiation .

FLUORESCENCE is instantaneous ,immediate phenomena of emitting visible light or radiation by a substance after absorbing visible or UV light.

1. Both the absorption and emission wavelengths are available .

2. Interferences may be avoided.

3. Data are not subjected to the relative error curve .

4. Accuracy is maintained at 99.999% transmittance range.

Fluorescence emission spectra arise due to transition from the free excited state to the ground singlet state .

Three states involves in luminescence

1. Ground singlet state. 2. Excited single state.(responsible for

fluorescence) 3. Excited triple state.( responsible for

phosphorescence).

MODIFIED DIAGRAMME SIMPLIFIED DIAGRAMME

Nature of molecule. Nature of substituents. Concentration. Adsorption. Light. Method of illumination. Oxygen. pH Photodecomposition. Temperature and viscosity.

F = 2.303 K I0 abc Where , F = intensity of fluorescent radiation. K = proportionality constant. I0 = intensity of incident radiation. a = absorptivity. b = length of cell path. C = concentration of absorbing

substances.

SINGLE BEAM FLUORIMETER DOUBLE BEAM FLUORIMETER

Determination of URANIUM,RUTHENIUM, CADMIUM.

Fluorescent indicators – Eosin, Fluorescein etc.

Fluorometric agent. Organic analysis. Pharmaceutical chemistry –

determination of Aneurine.

DETERMINATION OF VITAMIN B1 – Thiamine is non fluorescent but its oxidation product thiochrome fluorescence with blue colour.

DETERMINATION OF VITAMIN B2 – The solution is oxidised and fluorescence is measured by residual technique.

FLUORESCENCE MICROSCOPY. FLUORESCENCE LIFETIME IMAGING

MICROSCOPY. STUDY OF PROTIENS. BIOSENSOR. CELL SORTING. FORENSIC USE.

VALID FOR CONCENTRATION <10-5 M.- At high concentration self quenching takes place.

SUSCEPTIBLE TO INNERFILTER EFFECTS.- If emitted light is absorbed in the sample it cannot reach the detector and again the apparent fluorescence will decrease.

Fluorescence methods have greater selectivity and specificity than absorptiometric methods.

Not only in field of pharmaceutical also in field of animation , electronics , biologics fluorescence technique as well as fluorimetry is used .

1. INSTRUMENTA METHODS OF CHEMICAL ANALYSIS – Gurudeep R. chatwaal , Sham K. Anand ,Ch – 16, Pg . no. – 2.399 – 2.416.

2. A TEXT BOOK OF PHARMACEUTICAL ANALYSIS – Kenneth A. Connors , 3rd ed. , Ch – 9, Pg . no . – 235 -247.

3. INSTRUMENTAL METHODS OF ANALYSIS – Hobart H. Williard , Lynne L . Merritt, jr. , John A. Dean , Frank A. Settle, jr. Ch – 8 , Pg. no. – 197 -223 .

4. PRACTICAL PHARMACEUTICAL CHEMISTRY – A. H. beckett , J. B. Stenlake, 4th ed. , part two, Ch – 9 , Pg. no. – 358 – 378.

5 . ANALYTICAL BIOCHEMISTRY , Vol – 366 , issue 2 ,15 th july 2007 , pg no. – 149 – 155.

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