Emission Spectroscopy

Done by :Samyah Alanazi

Luminescence
Luminometry is the technique used to measure luminescence . Lluminescence is the emission of electromagnetic radiation in the energy range of visible light as a result of a reaction.

1- Chemiluminescence
*It arises from the relaxation of excited electrons transitioning back to the ground state. E.g. the reaction of luminol with oxygen produce 3aminophthalate which possesses a fluorescence spectrum of the product of the chemical reaction. *In this reaction, the resulted emission in the range of 400 to 450 nm.

The low photon yield of this reaction has: 1- limited its sensitivity 2- limit its application. However this problem tackled by adding enhancer molecules (luciferin, 6hydroxybenzothiazole) to the reaction in the present of peroxidase . As a result, the reaction can be followed for many minutes (30 or more) with a several thousand- fold increase in photon output .

Advantages and disadvantages

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

2- Bioluminescence
It describes the same phenomenon , only the reaction leading to fluorescent product is an enzymatic reaction. The most commonly used enzyme is Luciferase. Bioluminescence is a highly sensitive method, due to the high quantum yield of the underlying reaction . Some luciferase system work 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-magnatic radiation with matter which depend on properties of radiation and properties of the matter (sample structure).

3- Electrochemiluminescence
Its a process that based on the formation of an excited-state chemical intermediate that returns to the ground state by emitting photon . This is different from those in which an excited state is achieved by absorption of a photon. In this case the excited state achived by chemical reaction .

 1- Ru (complex) 2+ (electrode) = e- + Ru (complex)3+. 2- TPA (electrode) = e- + TPA *+ =TPA* + H+. 3- Ru (complex)3+ + TPA*+ e- = TPA degradation products + excited Ru (complex)2+. 4- excited Ru (complex)2+ = Ru (complex) 2+ +hv (light at 620 nm).

Instrumenta4on
Since no electromagnetic radiation is required as a source of energy for excitation, no light source and monochromator are required .Luminometry can be performed with a rather simple set-up, where a reaction is started in a cuvette or mixing chamber, and the resulting light is detected by a photometer. Photo-multiplier tube is needed to amplify the output signal prior to recording. Temperature must be controlled why??

Applica4ons
Chemiluminescence: (luminol) 1- Competitive binding assays. 2- phagocytosis. 3- Detect molecules and compounds with high efficiency.

Bioluminescence: (luciferase): 1- Determine concentration of ATP . 2- Determination of electron transfer cofactor.

Flourometry
Fluorescence is an emission phenomenon where an energy transition from a higher to lower state is accompanied by energy. Only molecules in their excited state are able to emit fluorescence: thus they have to be brought into a state of higher energy prior to the emission phenomenon. Once the molecule absorbs a photon, the molecule energy is greater than that of its environment, it seeks to eject the excess energy. When the energy is lost as an ejected photon, the result I fluroscence or phosphorescence emission.

Instrumenta4on

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.

 Standardization is usually not done by absolute procedure as in absorption spectroscopy 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.

 The emission of light usually varies on daily basis due to any change in the pH, temp. and solvent. For fluorometric assay zero only used for setting reagent blank. No equivalent to 100% scale of transmission. Absorbance of 0.1 is only allowed for standards to form a curve.

 Fluorescence attenuation assays : a constant amount of fluorescent dye is placed in each test and control solution. In test solution, the analyte cause reaction in which light absorbing compound is produced . The greater the amount of colored reaction product formed by analyte, the smaller amount absorbed by the fluorescent dye.

Time- delayed uorescence

- 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, stop illumination and measure the emitted fluroscence over a specified time from 400 microsec. To 800 microsec. After illumination. A limitation of this procedure is the need for separation steps because the chalets can not be measured in body fluids.

Fluorescence polariza4on
When a fluorescent molecule is excited with plane polarized light, light is emitted in the same polarized plane, provided that the molecule remains stationary throughout the excited state (which has a duration of 4 nanoseconds for fluorescein). If the molecule rotates and tumbles out of this plane during the excited state, light is emitted in a different plane from the excitation light. If vertically polarized light is exciting the fluorophore, the intensity of the emitted light can be monitored in vertical and horizontal planes (degree of movement of emission intensity from vertical to horizontal plane is related to the mobility of the fluorescently labeled molecule). If a molecule is very large, little movement occurs during excitation and the emitted light remains highly polarized. If a molecule is small, rotation and tumbling is faster and the emitted light is depolarized relative to the excitation plane.

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

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

Advantages and disadvantages

Advantages: Flourscence polarization measurments can be maid very accurately because they are less affected by variations in fluroscence measurments.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.