Fluorimetry

Views:
 
Category: Entertainment
     
 

Presentation Description

No description available.

Comments

Presentation Transcript

Seminar on Spectrofluorimetry :

Seminar on Spectrofluorimetry By Dr.Nalini Kanta Sahoo Associate Professor YalamartyPharmacy College Tarluwada,Anandapuram , Visakhapatnam,Anandapuram,530052

PowerPoint Presentation:

Introduction Definition Principle Instrumentation Applications CONTENTS

PowerPoint Presentation:

Introduction Absorption of UV/Visible radiation causes transition of electrons from ground state( lowenergy ) to excited state (highenergy). As excited state is not stable, excess energy is lost by , Collisional deactivation Emission of radiation (PhotoLuminescence). Emission Spectroscopy: Emission of radiation is absorbed. It is called luminescence. It is of 2two types LUMINESCENCE SPECTROSCOPY The emission of radiation from a species after that species has absorbed radiation.

fluorescence:

fluorescence When a beam of light is incident on certain substances they emit visible light or radiations. This is known as fluorescence. Fluorescence starts immediately after the absorption of light and stops as soon as the incident light is cut off. The substances showing this phenomenon are known as flourescent substances .

PowerPoint Presentation:

Photo luminiscence : Light without heat or coldlight Basically of 2 types Fluorescence: Part of energy is lost due to vibrational transitions and remaining energy is emitted as uv /visible radiation of longer wavelength incident light. Phosphorescence: Under favorable conditions, excited singlet state undergo transition to triplet state. Emission of radiation when electrons undergo transition from tr iplet state to ground state.

PowerPoint Presentation:

Understanding the terms…….. Singlet ground state: state in which electrons in a molecule are paired. Singlet excited state: state in which electrons are unpaired but of opposite spins. Triplet state: state in which unpaired electrons of same spin are present. Excitation process: absorption of energy or light followed by conversion from ground state to excite state. Relaxation process: process by which atom or molecule losses energy & returns to ground state. Most molecules have an even number of electrons which means that all of their electrons in the ground state must be paired: Singlet state (M = 2•0 + 1);. all electrons in ground state paired. Doublet state : (M = 2•½ + 1 = 2)a free radical (substance that has an odd number of electrons);. electrons can have 2 orientations in the magnetic field-opposed to the field and aligned.

PowerPoint Presentation:

Triplet state: (M = 2•1 + 1 = 3), excited state in which excited electron spin is flipped so that the spins are parallel. Singlet state Triplet state Diamagnetic Paramagnetic Probable Less probable Lifetime 10 -8 -10 -13 sec Lifetime up to ≥1s ENERGY LEVEL DIAGRAM : Let ground , the first excited, second excited etc. electronic states be: S 0 , S 1 , S 2 and etc..for all of the possible singlet states. Triplet states would then be T 1 , T 2 and so on . All electronic state has several vibrational and rotational states. Let ground , the first excited, second excited etc. electronic states be: S 0 , S 1 , S 2 and etc..for all of the possible singlet states.

PowerPoint Presentation:

Decay Processes: Internal conversion Movement of electron from one electronic state to another without emission of a photon, e.g. S 2 ® S 1 ) lasts about 10  12 sec. Predissociation internal conversion electron relaxes into a state where energy of that state is high enough to rupture the bond. Vibrational relaxation (10  10 -10  11 sec)- Energy loss associated with electron movement to lower vibrational state without photon emission. Intersystem crossing: Conversion from singlet state to a triplet state. e.g. S 1 to T 1 External conversion is a non- radiative process in which energy of an excited state is given to another molecule (e.g. solvent or other solute molecules). Related to the collisional frequency of excited species with other molecules in the solution. Cooling the solution minimizes this effect.

PowerPoint Presentation:

Fluorescence spectroscopy  : It is a type of electromagnetic spectroscopy which analyzes  fluorescence from a sample. It involves using a beam of light, usually  ultraviolet light , that excites the electrons in  molecules  of certain compounds and causes them to emit light of a lower energy, typically, but not necessarily,  visible light . A complementary technique is  absorption spectroscopy .

PowerPoint Presentation:

Classsfication : Based on the wavelength of emitted radiation when compared to absorbed radiation Stokesfluorescence : Wavelength of emitted radiation is longer than absorbed radiation Antistokes’s fluorescence: Wavelength of emitted radiation is shorter than absorbed radiation. Resonance fluorescence: Wavelength of emitted radiation is equal to that of absorbed radiation .

PowerPoint Presentation:

Quantum Efficiency Fluorescence, phosphorescence and internal conversion are competing processes. The fluorescence quantum efficiency and the phosphorescence quantum efficiency are defined as the fraction of molecules which undergo fluorescence and phosphorescence respectively.

PowerPoint Presentation:

Nature of substituent Effect of concentration Adsorption, Light Oxygen,PH Temp . &viscosity Intensity of incident light Path length Nature of molecule

PowerPoint Presentation:

Nature of molecules All the molecules cannot show the phenomenon of fluorescence. Only the molecules absorbs uv /visible radiation can show this phenomenon. Greater the absorbency of the molecule the more intense its fluorescence.

PowerPoint Presentation:

nature of substituent Electron donating group enhances fluorescence – e.g.:NH 2 ,OH etc. Electron withdrawing groups decrease or destroy fluorescence. e.g.:COOH,NO 2 , N= N etc. High atomic no: atom introduced into  electron system decreases fluorescence.

PowerPoint Presentation:

Fluorescence is directly proportional to concentration. FI = Q X I a i.e , F = QI O act Q = Constant for a particular substance I O = Constant for an instrument a = Molecular extinction coefficient t = Path length C = Concentration of the substance F = KC Where K represents all constants FI α Concentration.

PowerPoint Presentation:

Extreme sensitiveness of the method requires very dilute solution. Adsorption of the fluorescent substances on the container wall create serious problems. Hence strong solutions must be diluted.

PowerPoint Presentation:

Monochromatic light is essential for the excitation of fluorescence because the intensity will vary with wavelength. OXYGEN The presence of oxygen may interfere in 2 ways. 1] by direct oxidation of the fluorescent substances to non fluorescent. 2] by quenching of fluorescence.

PowerPoint Presentation:

Alteration of the pH of the solution will have significant effect on fluorescence. Fluorescent spectrum is different for ionized and un-ionized species. TEMPERATURE & VISCOSITY Increase in temperature/decrease in viscosity will decrease fluorescence.

PowerPoint Presentation:

The effective path length depends on both the excitation and emission slit width. Use of microcuvette does not reduce the fluorescence. Use of microcell may reduce interferences and increases the measured fluorescence

PowerPoint Presentation:

Decrease in fluorescence intensity due to specific effects of constituents of the solution. Due to concentration, pH, pressure of chemical substances, temperature, viscosity, etc. Types of quenching Self quenching Chemical quenching Static quenching Collision quenching

PowerPoint Presentation:

Fluorescence Concentration of fluorescing species Deviations at higher concentrations can be attributed to self-quenching or self-absorption . Fluorescence Concentration of fluorescing species Calibration curve (Low con) calibration curve (High con)

PowerPoint Presentation:

Here decrease in fluorescence intensity due to the factors like change in pH,presence of oxygen, halides &heavy metals. pH - aniline at pH 5-13 gives fluorescence but at pH <5 &>13 it does not exhibit fluorescence. halides like chloride,bromide,iodide & electron withdrawing groups like NO2,COOH etc. leads to quenching. Heavy metals leads to quenching, because of collisions of triplet ground state.

PowerPoint Presentation:

This occurs due to complex formation. e.g.. caffeine reduces the fluorescence of riboflavin by complex formation. COLLISIONAL QUENCHING It reduces fluorescence by collision. where no. of collisions increased hence quenching takes place.

PowerPoint Presentation:

INSTRUMENTATION

PowerPoint Presentation:

SOURCE OF LIGHT FILTERS AND MONOCHROMATORS SAMPLE CELLS DETECTORS

PowerPoint Presentation:

Power supply Source Excitation monochromator Emission monochromator Detector Sample cell Data processor

PowerPoint Presentation:

MERCURY ARC LAMP. XENON ARC LAMP. TUNGSTEN LAMP. TUNABLE DYE LASERS .

PowerPoint Presentation:

MERCURY ARC LAMP Produce intense line spectrum above 350nm. High pressure lamps give lines at 366,405, 436, 546,577,691,734nm. Low pressure lamps give additional radiation at 254nm.

PowerPoint Presentation:

Intense radiation by passage of current through an atmosphere of xenon. Spectrum is continuous over the range between over 250-600nm,peak intensity about 470nm .

PowerPoint Presentation:

Intensity of the lamp is low. If excitation is done in the visible region this lamp is used. It does not offer UV radiation.

PowerPoint Presentation:

FILTERS Primary filter-absorbs visible light & transmits uv light. Secondary filter-absorbs uv radiations & transmits visible light. MONOCHROMATORS Exitation monochromaters -isolates only the radiation which is absorbed by the molecule. Emission monochromaters -isolates only the radiation emitted by the molecule.

PowerPoint Presentation:

The majority of fluorescence assays are carried out in solution. Cylindrical or rectangular cells fabricated of silica or glass used. Path length is usually 10mm or 1cm. All the surfaces of the sample holder are polished in fluorimetry.

PowerPoint Presentation:

PHOTOVOLTAIC CELL PHOTO TUBE PHOTOMULTIPLIER TUBES – Best and accurate.

PowerPoint Presentation:

Multiplication of photo electrons by secondary emission of radiation. A photo cathode and series of dynodes are used. Each anode is maintained at 75-100v higher than the preceding one. Over all amplification of 10 6 is obtained.

PowerPoint Presentation:

SINGLE BEAM FLUORIMETER DOUBLE BEAM FLUORIMETER SPECTROFLUORIMETER(DOUBLE BEAM)

PowerPoint Presentation:

Tungsten lamp as source of light. The primary filter absorbs visible radiation and transmits uv radiation. Emitted radiation measured at 90 o by secondary filter. Secondary filter absorbs uv radiation and transmits visible radiation.

PowerPoint Presentation:

Simple in construction Easy to use. Economical disadvantages It is not possible to use reference solution & sample solution at a time. Rapid scanning to obtain Exitation & emission spectrum of the compound is not possible.

PowerPoint Presentation:

Similar to single beam instrument. Two incident beams from light source pass through primary filters separately and fall on either sample or reference solution. The emitted radiation from sample or reference pass separately through secondary filter.

PowerPoint Presentation:

Sample & reference solution can be analyzed simultaneously. disadvantage Rapid scanning is not possible due to use of filters.

PowerPoint Presentation:

Advantages : More sensitive when compared too the radiation absorption techniques. Concentrations as low as μ g/ml or ng /ml can be determined. Precision up to 1% can be achieved easily. As both excitation & emission wavelengths are characteristicitis more specific than absorption methods.

PowerPoint Presentation:

Applications Determination of Organic substances , Plant pigments ,steroids ,proteins , naphthols etc can be determined at low concentrations . Generally used to carry out qualitative as well as quantitative analysis for a grea taromatic compounds present in cigarette smoking , airpollutant concentrates & automobile exhausts. Atomic Fluorescence Spectroscopy (AFS) techniques are useful in other kinds of analysis/measurement of a compound present in air or water, or other media, such as  CVAFS (Cold vapour atomic fluorescence spectroscopy)  which is used for heavy metals detection, such as mercury.

PowerPoint Presentation:

Determination of inorganic substances, Determination of vitaminB1 (thiamine )in food samples like meat,cereals etc. Determination of VitaminB2(riboflavin) .This method is generally used to measure the amount of impurities present in the sample. Extensively used in the field of nuclear research for the determination of uranium salts. Most important applications are found in the analyses of food products, pharmaceuticals ,clinical samples and natural products .

PowerPoint Presentation:

Fluorescent indicators: Intensity and colour of the fluorescence of many substances depend upon the pH of solutions. These are called as forescent indicators and are generally used in acidbase titrations . Eg : Eosin–pH3. 0-4.0–colour less to green Fluorescien–pH4.0-6.0– colourless to green

PowerPoint Presentation:

Douglas A Skoog , Principles of instrumental analysis H:\UV-Vis Luminescence Spectroscopy - Theory.mht Dr.B.K.Sharma , Instrumental methods of chemical analysis Gurdeep R Chatwal , Instrumental methods of chemical analysis

PowerPoint Presentation:

http://images.google.co.in/imghp?oe=UTF-8&hl=en&tab=wi&q=fluorescence http://en.wikipedia.org/wiki/Fluorescence http://www.bertholdtech.com/ww/en pub/bioanalytik/biomethods/fluor.cfm

PowerPoint Presentation:

Thank u all….. Have a great day

authorStream Live Help