Luminometry and flurometr.pdf

7/28/2019 Luminometry and flurometr.pdf

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Emission Spectroscopy

Done by :Samyah Alanazi


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Luminescence

Luminometry is the technique used tomeasure luminescence .

Lluminescence is the emission ofelectromagnetic radiation in the energy

range of visible light as a result of a

reaction.


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1- Chemiluminescence

*It arises from the relaxation of excited electronstransitioning back to the ground state.

E.g. the reaction of luminol with oxygen produce 3-aminophthalate which possesses a fluorescencespectrum of the product of the chemical reaction.

*In this reaction, the resulted emission in the rangeof 400 to 450 nm.


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The low photon yield of this reaction has:

1- limited its sensitivity

2- limit its application.

However this problem tackled by addinge n h a n c e r m o l e c u l e s ( l u c i f e r i n , 6 -hydroxybenzothiazole) to the reaction in thepresent of peroxidase . As a result, thereaction can be followed for many minutes (30or more) with a several thousand- fold increasein photon output .


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Advantagesanddisadvantages

Advantage: very sensitive. Disadvantage: Reaction performed in a heterogeneous

system.


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2- Bioluminescence

It describes the same phenomenon , only the reaction leading tofluorescent product is an enzymatic reaction. The most commonly

used enzyme is Luciferase.

Bioluminescence is a highly sensitive method, due to the highquantum yield of the underlying reaction . Some luciferase systemwork with almost 100% efficiency . For comparison, the

incandescent light bulb loses about 90% of the input energy to heat.

Because Luminescence does not depend on any optical excitation,problems with auto fluorescence in assays are eliminated.

quantum phenomenon: the interaction of electro-magnaticradiation with matter which depend on properties of radiation and

properties of the matter (sample structure).


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3-Electrochemiluminescence

Its a process that based on the formationof an excited-state chemical intermediate

that returns to the ground state by emitting

photon . This is different from those inwhich an excited state is achieved by

absorption of a photon. In this case the

excited state achived by chemicalreaction .


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1- Ru (complex) 2+ (electrode) = e- + Ru(complex)3+.

2- TPA (electrode) = e- + TPA *+ =TPA* +H+.

3- Ru (complex)3+ + TPA*+ e- = TPAdegradation products + excited Ru

(complex)2+. 4- excited Ru (complex)2+ = Ru (complex)

2+ +hv (light at 620 nm).


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Instrumenta4on

Since no electromagnetic radiation isrequired as a source of energy for excitation,no light source and monochromator are

required .Luminometry can be performed witha rather simple set-up, where a reaction isstarted in a cuvette or mixing chamber, andthe resulting light is detected by a

photometer. Photo-multiplier tube is neededto amplify the output signal prior to recording.Temperature must be controlled why??


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Applica4ons

Chemiluminescence: (luminol) 1- Competitive binding assays. 2- phagocytosis. 3- Detect molecules and compounds withhigh efficiency. Bioluminescence: (luciferase): 1- Determine concentration of ATP . 2- Determination of electron transfer co-

factor.


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Flourometry

Fluorescence is an emission phenomenon wherean energy transition from a higher to lower state isaccompanied by energy. Only molecules in theirexcited state are able to emit fluorescence: thus

they have to be brought into a state of higherenergy prior to the emission phenomenon.

Once the molecule absorbs a photon, themolecule energy is greater than that of its

environment, it seeks to eject the excess energy.When the energy is lost as an ejected photon, theresult I fluroscence or phosphorescence emission.


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Instrumenta4on


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Limita4ons

1- compound signals are affected by; 1- solvent.2- pH

3- Temperature. 4- absorbance of the solution 5-presence of interfering or quenching

compound.


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Standardization is usually not done byabsolute procedure as in absorptionspectroscopy because fluorescence varies

depend on: 1- intensity of Io light on the sample. 2- the amounted of light interceptedby the

detectoras controlled by slits. 3- the band width of light analysed. 4- the efficiency of the detector.


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The emission of light usually varies ondaily basis due to any change in the pH,

temp. and solvent.

For fluorometric assay zero only used forsetting reagent blank. No equivalent to

100% scale of transmission.

Absorbance of 0.1 is only allowed forstandards to form a curve.


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Fluorescence attenuation assays : aconstant amount of fluorescent dye is

placed in each test and control solution. In

test solution, the analyte cause reaction inwhich light absorbing compound is

produced . The greater the amount of

colored reaction product formed byanalyte, the smaller amount absorbed by

the fluorescent dye.


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Time-delayedfluorescence

- done to improve sensitivity of the technique. - it will increase the specificity of analysis. - specialized instruments used this technique

to illuminate the sample for a time, stopillumination and measure the emittedfluroscence over a specified time from 400microsec. To 800 microsec. After illumination.

A limitation of this procedure is the need forseparation steps because the chalets can notbe measured in body fluids.


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Fluorescencepolariza4on When a fluorescent molecule is excited with plane polarized

light, light is emitted in the same polarized plane, providedthat the molecule remains stationary throughout the excitedstate (which has a duration of 4 nanoseconds for fluorescein).

If the molecule rotates and tumbles out of this plane duringthe excited state, light is emitted in a different plane from theexcitation light. If vertically polarized light is exciting thefluorophore, the intensity of the emitted light can be monitoredin vertical and horizontal planes (degree of movement ofemission intensity from vertical to horizontal plane is related to

the mobility of the fluorescently labeled molecule). If amolecule is very large, little movement occurs duringexcitation and the emitted light remains highly polarized. If amolecule is small, rotation and tumbling is faster and theemitted light is depolarized relative to the excitation plane.


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P= Ivv Ihv /Ivv+Ihv. Ihv ... Intensity with polarizers parallel.Ivv ... Intensity with polarizersperpendicular.


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Factors affecting final polarization are: 1- viscosity.2- Size of molecule.


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Advantages and disadvantages

Advantages: Flourscence polarization measurments can be

maid very accurately because they are lessa f f ec ted by va r i a t i ons i n f l u roscencemeasurments.Thus precision is readily achieved.

Disadvantages: Is limited to assays that can use fluoroscence dye. Less flexible than absorption spectroscopy. Crucial to control viscosity and temp.

Luminometry and flurometr.pdf

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