FLUORIMETRYand

Phosphorimetry g. Enosh

2016672003

FLUORIMETRY

It is measurement of fluorescence intensity at a particular wavelength with the help of a filter fluorimeter or a spectrofluorimeter.

Phosphorimetry

A form of fluorimetry in which phosphorescence of a sample is measured in a conjunction with a pulsed sourse of radiation.

Photoluminesence“Photoluminescence (PL) is the spontaneous emission of light from a material under optical excitation.” It is futher subdivided into two types

a. Flourescenceb. Phosphorescence

FluorescenceWhen a beam of light is incident on certain substances they emit visible light or radiation .This phenomenon is called fluorescence. Substances showing this phenomenon are fluorescent substances.This phenomenon is Fluorescence instantaneous i.e start emitting radiation immediately after the absorption and stops when incident light is cut off.Fluorescent substances emit maximum radiation with in 10-10 to 10-8sec of absorption. This is also called delayed fluorescence

PhosphorescenceWhen light is incident on certain substances ,they emit light continuously even after the light is cut off . This is called phosphorescence and such substances are called as phosphorescent substances.In the fluorescence process, the electron did not change its spin direction But under the appropriate conditions, a spin-flip can occurThe light emission process must wait until electron undergoes a spin-flip to revert back to it original statePhosphorescent substances emit maximum radiation within 10-4 to 20 sec or longer.

Difference between Phosphorescence and fluorescence

Phosphorescence

Fluorescence

The emission could proceed either from a singlet or triplet state.

The emission could proceed only from a singlet state.

short-live electrons (<10-5 s) in the excited state of fluorescence

longer lifetime of the excited state (second to minutes)

CONTENTS•Principle•Factors effecting Fluorescence,Phosphorescence•Intensity•Instrumentation•Applications•Conclusion

PRINCIPLE•Molecule contains electrons, electrons and non bonding (n) electron. •The electrons may be present in bonding molecular orbital. •It is called as highest occupied molecular orbital (HOMO).•It has lest energy and more stable.•When the molecules absorbs radiant energy from a light source, the bonding electrons may be promoted to anti bonding molecular orbital. It has more energy and hence less stable.

The process of promotion of electrons from HOMO to LOMO with absorption of energy is called as excitation.Singlet state:- a state in which all the electrons in a molecule are paired Doublet state:- a state in which un paired electrons is present or Triplet state:- a state in which unpaired electrons of same spin present Singlet excited state:- a state in which electrons are unpaired but of opposite spin like (un paired and opposite spin)

When light of appropriate wavelength is absorbed by a molecule the electrons are promoted from singlet ground state to singlet excited state. Once he molecule is in this excited state relaxation can occur via several process like emission of radiation.

The process can be the following:-

1) Collision deactivation 2)Fluorescence

3)Phosphorescence.

Collisional de activation :-

In which entire energy lost due to collision de activation and no radiation emitted.Fluorescence:-

Excited singlet state is highly unstable. Relaxation of electrons from excited singlet to singlet ground state with emission of light.Phosphorescence:-

At favorable condition like low temperature and absence of oxygen there is transition from excited singlet state to triplet state which is called as intersystem crossing . The emission of radiation when electrons undergo transition from triplet state to singlet ground state.

Factors effecting fluorescence intensity

1. Concentration2. Quantum yield of fluorescence3. Intensity of incident light4. Oxygen5. Ph6. Temperature& viscosity7. Photodecomposition8. Quenchers9. Scatter10. Solvent

Concentration:-•Fluorescence intensity is proportional to concentration of substance only when the absorbance is less than 0.02

Beer-Lambert Law

A = log(I /I 0) = €.c.lI = transmitted light intensity Io = incident light intensity c = concentration (M) l = path length of cell (cm) €= molar extinction coefficient (usually in the range 0-105 , with <103 considered to be a week absorption

QUANTUM YIELD OF FLUORESCENCE:-() NUMBER OF PHOTONS EMITTED () = ———————————

NUMBER OF PHOTONS ABSORBEDS

It Is Always Less Than 1.0 Since Some Energy Is Lost By Radiation less Pathways (Collisions, Intersystem Crossing, Vibrational Relaxation)

INTENSITY OF INCIDENT LIGHT:-

Increase In The Intensity Of Incident Light On The Sample Fluorescence Intensity Also Increases effect.

Dissolved oxygen•Oxygen with unpaired electrons dramatically decrease fluorescence and cause interference in fluorimetric determinations.•The paramagnetic nature of molecular oxygen promotes intersystem crossing from singlet to triplet states in other molecules.•The longer lifetimes of the triplet states increases the opportunity for radiation less deactivation to occur.

PH•Relatively small changes in pH can sometimes cause substantial changes in the fluorescence intensity and spectral characteristics of fluorescence.

Temperature•A rise in temperature is almost always accompanied by a decrease in fluorescence.•The change in temperature causes the viscosity of the medium to change which in turn changes the number of collisions of the molecules of the fluorophore with solvent molecules. •The increase in the number of collisions between molecules in turn increases the probability for deactivation by internal conversion and vibrational relaxation.

Photochemical decomposition:-Absorption of intense radiation leads to photochemical decomposition of a fluorescent substance to less fluorescent or non fluorescent substance.

Quenchers:-Quenching is the reduction of fluorescence intensity by the presence of substance in the sample other than the fluorescent analyte.

SCATTER

•Scatter is mainly due to colloidal particles in solution

•Scattering of incident light after passing through the sample leads to decrease in fluorescence intensity.

•A higher fluorescence is observed when the solvents do not contain heavy atoms while phosphorescence increases due to the presence of heavy atoms in the solvent.

Solvent•The changes in the “polarity” or hydrogen bonding ability of the solvent may also significantly affect the fluorescent behaviour of the analyte.•The difference in the effect of solvent on the fluorescence is attributed to the difference in their ability to stabilise the ground and excited states of the fluorescent molecule.•Increased viscosity increases fluorescence as the deactivation due to collisions is lowered.

INSTRUMENTATION

INSTRUMENTATION

•SOURCE OF LIGHT

•FILTERS AND MONOCHROMATORS

•SAMPLE CELLS

•DETECTORS

1)SOURCE OF LIGHT:-Mercury vapour lamp: Mercury vapour at high pressure give intense lines on continuous background above 350nm.low pressure mercury vapour gives an additional line at 254nm.it is used in filter fluorimeter.

Xenon arc lamp: It give more intense radiation than mercury vapour lamp. it is used in spectrofluorimeter.

Tungsten lamp:- If excitation has to be done in visible region this can be used. It is used in low cost instruments.

mercury vapour lamp

xenon arc lamp

tungsten lamp

2) FILTERS AND MONOCHROMATORS:-

Filters: These are nothing but optical filters works on the principle of absorption of unwanted light and transmitting the required wavelength of light. In inexpensive instruments fluorimeter primary filter and secondary filter are present.•Primary filter:- Absorbs visible radiation and transmit UV radiation.•Secondary filter:-Absorbs UV radiation and transmit visible radiation.

Monochromators:

They convert polychromatic light into monochromatic light. They can isolate a specific range of wavelength or a particular wavelength of radiation from a source. •Excitation monochromators:-provides suitable radiation for excitation of molecule.

•Emission monochromators:- isolate only the radiation emitted by the fluorescent molecules.

3) Sample cells These are ment for holding liquid samples. These are made up of quartz and can have various shapes ex: cylindrical or rectangular etc.

4) Detectors

Photometric detectors are used they are •Barrier layer cell/Photo voltaic cells•Photomultiplier cells

INSTRUMENTS

The most common types are:-

•Single beam (filter) fluorimeter

•Double beam (filter )fluorimeter

•Spectrofluorimeter(double beam

Single beam filter fluorimeter

•It contains tungsten lamp as a source of light and has an optical system consists of primary filter.

•The emitted radiations is measured at 900 by using a secondary filter and detector. Primary filter absorbs visible radiation and transmit uv radiation which excites the molecule present in sample cell.

Cont.•In stead of 900 if we use 1800 geometry as in colorimetry secondary filter has to be highly efficient other wise both the unabsorbed uv radiation and fluorescent radiation will produce detector response and give false result.

•Single beam instruments are simple in construction cheaper and easy to operate.

In double beam fluorimeter:-

It is similar to single beam except that the two incident beams from a single light source pass through primary filters separately and fall on the another reference solution. Then the emitted radiations from the sample or reference sample pass separately through secondary filter and produce response combinly on a detector.

Spectrofluorimeter:

•In this primary filter in double beam fluorimeter is replaced by excitation monochromator and the secondary filter is replaced by emission monochromator.

•Incident beam is split into sample and reference beam by using beam splitter.

Single beam (filter) fluorimeter

Double beam (filter )fluorimeter

Spectrofluorimeter(double beam

Phosphorimetry

•Instrumentation

•Instrument

•Applications

Instrumentation• Excitation Source• Filters/Monochromators for excitation

radiations• Phosphorscope(sample Cell)

Excitation Source

High intensity source of UV light are usedLasers – A laser makes it possible to have narrow wavelength intervals that offer very high energy irradiation. This is useful when a large amount of energy is needed to produce the Phosphorescence in the sample.

Photodiodes – Photodiodes are specialized diodes that can be configured in a manner that allows electrons to flow towards the sample so that the excess energy excites the phosphorescent particles.

Xenon Arcs – Arcs of Xenon can produce the right amount of radiation for Phosphorescent materials.

Mercury Vapor – Since mercury vapor can create ultraviolet radiation when electrical current is passed through it, it is good for use with materials that shows Phosphorescence under the ultraviolet radiation.

note: Care should be taken as Intense UV light is hazardous for health

Filters and monochromators used in a phosphorimeter device.

• Filters– Absorption– Interfernce

• Monochromators It allow wavelength adjustment. Monochromators make it possible to do so with a

diffraction grating. Diffraction grating is an optical component with a periodic structure, which splits

and diffracts light into several beams travelling in different directions.

Cont.-The primary filters that excite the sample provide the appropriate wavelength -While the secondary filters monochromate the emitted light when sent to the detector.

Phosphoresence spectroscopy detectors may have a

single channel (single wavelength from sample)multiple channels (multiple wavelengths detection)

Instrument

phosphoroscope

A phosphoroscope is piece of experimental equipment devised  to measure how long it takes a phosphorescent material to stop glowing after it has been excited.

Phosphoroscope1.The Becquerel or rotating disc

phosphoroscope• A rotating disk excitation optical chopper, with

three open and three larger opaque areas, is used to alternately excite the sample and allow phosphorescence to be measured.

• By measuring the phosphorescence intensity at several time intervals along the emission decay curve, a recorder trace of the decay with respect to time can be produced.

Cont.The analytical precision and accuracy for quantitative measurements is improved by rotating the sample tube. This minimizes variation in the signal due to sample in homogeniety resulting from imperfect glass formation at low-temperatures .

2.The Rotating-Can Phosphoroscope:• It consists of hollow cylinder having one or more

slit which are equally spaced in the circumference.• This is rotated by a variable-speed

motor(>1000rpm)• when the rotating-can is rotated by a motor the

sample is excited show fluorescence and phosphorescence

For Fluorescence the emission monochromator is blocked by the can so that fluorescence and scattered radiation cannot be detected.

Cont.• As the can rotates, the excitation beam is blocked and

the fluorescence and scattered radiation decay to a negligible amount.

• Further rotation of the can will bring the window into alignment with the emission monochromator entrance slit and phosphorescence radiation will pass into the emission monochromator and onto the sample photomultiplier

Applications of Phosphorescence Spectroscopy

• Pharmaceutical Applications

• Clinical Applications

• Environmental Applications

• Entertainment Applications

Pharmaceutical Applications• The majority of phosphorescence applications

have been applied in the drug and pharmaceutical field and in the analysis of pesticides

• A number of the sulphonamide class of drugs exhibit phosphorescence as do cocaine, procaine, chlorpromazin and salicylic acid.

Clinical Applications• The phosphorescence intensity of the rare

earths increases tremendously when they are covalently bound to certain molecules and this feature has been used in the analysis of transferin in blood

• dual-wavelength phosphorimeter used to measure microvascular PO2 (µPO2) in different depths in tissue and demonstrates its use in rat kidney.

Environmental Applications

•Phosphorescence has been used in the detection•In air and water-borne pollutantsfor the analysis of impurities in polycyclic aromatic hydrocarbons and in petroleum products.

Entertainment Applications

•Use of phosphor coated stamps and envelopes has appeared which gives a fascinating insight into the practical use of phosphorescence.

•They used phosphors in lamps

•TV tubes.

Advantages SENSTIVITY: This technique can measure

concentration as low as microgram/ml or even ng/ml.

SPECIFICITY: More specific than absorption technique.

PRECISION: Up to 100% can easily achieved in fluorimetery and Phosphorimetry

.

LimitationsChange in pH affects fluorescent and Phosphorimetry intensity.

Dissolved oxygen may affect fluorescent intensity.

Traces of halides, heavy metals, etc can affect fluorescent intensity.

Applications of photoluminescence methods1) Determination of uranium salt by flourimetry.2) Determination of inorganic species -by direct method involve formation of fluorescing

chelates. eg. aromatic structure with two aromatic donor

function group that permit chelate formation with metal ion decrease in fluorescing emission because of quenching

Cont.3) Organic analysis- (adenine, antranilic acid, uric acid, morphine, proteins etc..)4) Determination of aspirin in blood serum with high sensitivity by phosphometric at liq. Nitrogen temperature. - low conc. of procaine, cocaine, chlorpromazine can be determined. 5 Determination of Vit B1(Thiamine). - determination of Vit B2(riboflavin)

Comparison of fluorimetry and phosphorimetry with absorption

methods.

Sensitivity of luminescence methods is generally 10 to 103 times greater than sensitivity of absorption method.

Luminescence method have greater specificity and selectivity than absorption methods.

References :•PhosphorimetryR. J. Keirs, R. D. Britt, W. E. Wentworth

• Phosphorimetry : theory, instrumentation, and applicationsRobert J. Hurtubise.

•Principles of Fluorescence SpectroscopyAuthors: Lakowicz, Joseph R.

• Google Scholar and Google images.

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