CC LEC M3 U2

• A fluorometer is a device that measures the fluorescence of a sample, where fluorescence is the emission of light by a substance that has absorbed light
• Fluorometers are used for various applications including detecting and quantifying fluorescent molecules, studying the structure and function of proteins, measuring the activity of enzymes, and screening for drugs
• Module objectives for Clinical Chemistry 1 include differentiating the methods of instrumentation based on their principle of operation, components, advantages, limitations, and application in measuring different analytes
• Another objective in Clinical Chemistry 1 is practicing appropriate preventive maintenance on different instruments used in clinical chemistry
• In Clinical Chemistry 1, there are various topics to cover like Spectrometry, Luminescence, Electro-analytical Methods, Chromatography, Automation, and Point of Care Testing
• Each topic in Clinical Chemistry 1 has activities like ENGAGE, EXPLORE, EXPLAIN, ELABORATE, and EVALUATE
• For each topic in Clinical Chemistry 1, students are required to read and engage with the content, explore further, explain the concepts, elaborate on the topic, and evaluate their understanding
• Property of and for the exclusive use of SLU: Reproduction, storing, distributing, or transmitting any part of the document without prior written permission is strictly prohibited
• Fluorometers measure the fluorescence of a sample, where fluorescence is the emission of light by a substance that has absorbed light
• Fluorometers are used for various applications, including detecting and quantifying fluorescent molecules, studying the structure and function of proteins, measuring the activity of enzymes, and screening for drugs
• Fluorescence is more sensitive than absorption, making it easier to detect luminescence from a small number of molecules in a sample
• Luminescence is any emission of light or radiant energy when an electron returns from an excited or higher energy level to a lower energy level
• For quantitative analysis, the intensity of luminescence is proportional to the concentration of the emitting species over a limited concentration range and the incident radiant power
• The components of a fluorometer include a light source, excitation monochromator, sample cell, emission monochromator, and detector
• Excitation sources for fluorometers include xenon, quartz-halogen, mercury arc lamps, and lasers
• The detector commonly used in fluorometers is the photomultiplier tube, providing a wide choice of spectral responses, rapid photon response time, and sensitivity
• Fluorescent assays are used to measure fluorescent tags or labels, not naturally occurring fluorescent molecules, and are applied in hematofluorometry and flow cytometry
• Factors influencing fluorescence measurements include concentration effects like inner-filter effect and concentration quenching, as well as light scattering
• A fluorometer measures the fluorescence of a sample, where fluorescence is the emission of light by a substance that has absorbed light

  
• Fluorometers are used for various applications, including detecting and quantifying fluorescent molecules, studying the structure and function of proteins, measuring enzyme activity, and screening for drugs
• Factors affecting fluorescence measurements include radiationless energy transfer, light scattering, solvent and cuvet effects, sample matrix effects, temperature effects, and photodecomposition
• Stoke’s Shift is the difference between the excitation energy and emitted fluorescence, and it is characteristic for a given molecular type
• Quenching by the solvent can lead to a loss of fluorescence in fluorophores due to energy transfer or other mechanisms
• Sample matrix effects in fluorescence measurements can be influenced by compounds like proteins and bilirubin in serum or urine samples, contributing to unwanted background fluorescence
• Temperature fluctuations can affect the fluorescence efficiency of compounds, with fluorescence intensity decreasing with increasing temperature
• In conventional fluorometry, intense light sources on weakly fluorescing solutions may cause the photochemical decomposition of the analyte
• Phosphorescence is luminescence that continues even after the radiation causing it has ceased, with a longer decay time than fluorescence
• Chemiluminescence and bioluminescence are types of luminescence caused by chemical or electrochemical reactions, with chemiluminescence involving compounds reacting with an oxidizing agent
• Bioluminescence is a special form of chemiluminescence occurring within biological systems, with enzymes like luciferase increasing the efficiency of luminescence
• Electrochemiluminescence differs from other luminescence types as reactive species are electrochemically generated from stable precursors at the surface of an electrode
• Light scattering techniques like turbidimetry and nephelometry are used to measure scattered light, with turbidimetry measuring the decrease in intensity caused by scattering, reflectance, and absorption
• Nephelometry detects light energy scattered or reflected toward a detector not in the direct path of transmitted light, with common nephelometers measuring scattered light at right angles to the incident light
• Nephelometers must ideally be free of stray light to ensure accurate measurements
• Luciferase in the bioluminescence reaction in fireflies acts as a catalyst
• In fluorescence, the emitted light is of less energy than the excitation light