Introduction, theoretical principle, quantum efficiency of fluorescence, molecular structure of
fluorescence, instrumentation, factors influencing the intensity of fluorescence, comparison of fluorometry with spectrophotometry, application of fluorometry in pharmaceutical analysis
This document provides an overview of infrared (IR) spectroscopy. It discusses the principle behind IR spectroscopy, the different modes of molecular vibration, instrumentation including sources, detectors and monochromators. It also covers sample handling techniques, factors that affect vibrational frequencies and applications of IR spectroscopy such as structure elucidation.
In molecular spectroscopy, a Jablonski diagram is a diagram that illustrates the electronic states of a molecule and the transitions between them. The states are arranged vertically by energy and grouped horizontally by spin multiplicity.
This document discusses Fourier transform nuclear magnetic resonance (FT-NMR) spectroscopy. It begins by introducing NMR spectroscopy and its ability to provide chemical structure information. It then explains that FT-NMR uses a pulse of radiofrequency energy to simultaneously excite all nuclei, followed by a Fourier transform to separate the signal into frequencies. This allows the full spectrum to be obtained within seconds, offering advantages over continuous wave NMR in speed, sensitivity, and ability to average multiple signal acquisitions to improve resolution. The document outlines the components of an FT-NMR spectrometer and factors that influence sensitivity.
This document discusses factors that affect fluorescence and phosphorescence. It defines fluorescence and phosphorescence as types of molecular luminescence that are excited by photon absorption. The main difference is that fluorescence involves no change in electron spin, while phosphorescence does involve a change. Several factors can influence emission, including molecular structure and rigidity, temperature, solvent properties, pH, dissolved oxygen, concentration, and the presence of heavy atoms. More rigid and planar structures favor fluorescence and phosphorescence. Higher temperatures, viscosities, and oxygen levels decrease emission, while appropriate solvent polarity and pH can increase it.
This document provides an overview of fluorometry, including basic concepts, instrumentation, and applications. It discusses how fluorescence occurs when a molecule absorbs light at one wavelength and reemits light at a longer wavelength. Factors that affect fluorescence such as temperature, pH, and dissolved oxygen are also covered. The relationship between fluorescence intensity and concentration is explained. Additionally, the document defines fluorescence polarization and describes various types of quenching including self-quenching, chemical quenching, and collisional quenching.
1) Protons experience different amounts of shielding depending on their chemical environment and electron densities around them.
2) The chemical shift value provides a number independent of the NMR instrument used to measure it.
3) Factors like electronegativity of nearby atoms, hybridization, hydrogen bonding, and anisotropic effects influence the chemical shift values of protons in a molecule.
Nuclear Magnetic Resonance (NMR) spectroscopy involves absorbing radio frequency radiation by atomic nuclei in a magnetic field. NMR can be used to study the magnetic properties and local chemical environments of different nuclei, deduce molecular structure, and identify atoms in neighboring groups. The number and positions of NMR signals provide information about the number of different proton types in a molecule and their magnetic shielding. Signal intensities correlate with proton numbers, and splitting patterns indicate neighboring protons. NMR has applications in materials science, chemical analysis, studying hydrogen bonding and drug design.
Fluorescence spectroscopy analyzes the fluorescent properties of molecules. It works by exciting a molecule to a higher electronic state using a photon, causing it to emit a photon of lower energy as it returns to the ground state. The difference in wavelengths allows detection of emission photons. Key aspects covered include the principles of absorption and emission, instrumentation used, and different types of data that can be recorded such as fluorescence measurements, steady state techniques, and fluorescence anisotropy/polarization.
This document provides an overview of infrared (IR) spectroscopy. It discusses the principle behind IR spectroscopy, the different modes of molecular vibration, instrumentation including sources, detectors and monochromators. It also covers sample handling techniques, factors that affect vibrational frequencies and applications of IR spectroscopy such as structure elucidation.
In molecular spectroscopy, a Jablonski diagram is a diagram that illustrates the electronic states of a molecule and the transitions between them. The states are arranged vertically by energy and grouped horizontally by spin multiplicity.
This document discusses Fourier transform nuclear magnetic resonance (FT-NMR) spectroscopy. It begins by introducing NMR spectroscopy and its ability to provide chemical structure information. It then explains that FT-NMR uses a pulse of radiofrequency energy to simultaneously excite all nuclei, followed by a Fourier transform to separate the signal into frequencies. This allows the full spectrum to be obtained within seconds, offering advantages over continuous wave NMR in speed, sensitivity, and ability to average multiple signal acquisitions to improve resolution. The document outlines the components of an FT-NMR spectrometer and factors that influence sensitivity.
This document discusses factors that affect fluorescence and phosphorescence. It defines fluorescence and phosphorescence as types of molecular luminescence that are excited by photon absorption. The main difference is that fluorescence involves no change in electron spin, while phosphorescence does involve a change. Several factors can influence emission, including molecular structure and rigidity, temperature, solvent properties, pH, dissolved oxygen, concentration, and the presence of heavy atoms. More rigid and planar structures favor fluorescence and phosphorescence. Higher temperatures, viscosities, and oxygen levels decrease emission, while appropriate solvent polarity and pH can increase it.
This document provides an overview of fluorometry, including basic concepts, instrumentation, and applications. It discusses how fluorescence occurs when a molecule absorbs light at one wavelength and reemits light at a longer wavelength. Factors that affect fluorescence such as temperature, pH, and dissolved oxygen are also covered. The relationship between fluorescence intensity and concentration is explained. Additionally, the document defines fluorescence polarization and describes various types of quenching including self-quenching, chemical quenching, and collisional quenching.
1) Protons experience different amounts of shielding depending on their chemical environment and electron densities around them.
2) The chemical shift value provides a number independent of the NMR instrument used to measure it.
3) Factors like electronegativity of nearby atoms, hybridization, hydrogen bonding, and anisotropic effects influence the chemical shift values of protons in a molecule.
Nuclear Magnetic Resonance (NMR) spectroscopy involves absorbing radio frequency radiation by atomic nuclei in a magnetic field. NMR can be used to study the magnetic properties and local chemical environments of different nuclei, deduce molecular structure, and identify atoms in neighboring groups. The number and positions of NMR signals provide information about the number of different proton types in a molecule and their magnetic shielding. Signal intensities correlate with proton numbers, and splitting patterns indicate neighboring protons. NMR has applications in materials science, chemical analysis, studying hydrogen bonding and drug design.
Fluorescence spectroscopy analyzes the fluorescent properties of molecules. It works by exciting a molecule to a higher electronic state using a photon, causing it to emit a photon of lower energy as it returns to the ground state. The difference in wavelengths allows detection of emission photons. Key aspects covered include the principles of absorption and emission, instrumentation used, and different types of data that can be recorded such as fluorescence measurements, steady state techniques, and fluorescence anisotropy/polarization.
This document provides an overview of fluorescence spectroscopy. It describes how luminescence occurs when a system absorbs external energy like light and emits photons. Specifically, fluorescence involves absorbing ultraviolet or visible light which causes molecule excitation, then reemission of light. The document outlines fluorescence instrumentation components like light sources, wavelength selection using filters or monochromators, detectors, and sample holders. It also discusses related topics such as phosphorescence, absorption spectra, and the advantages and disadvantages of fluorescence spectroscopy.
Phosphorescence is the process by which materials emit light after absorbing radiation or energy. It was first observed in the 17th century but not studied scientifically until the 19th century. Phosphorescent materials store absorbed energy and release it slowly as light over minutes or hours by trapping electrons in an excited state. There are two types of photoluminescence - fluorescence which emits light quickly, and phosphorescence which emits light slowly. Common phosphorescent materials include zinc sulfide and strontium aluminate.
1. Fluorescence is the emission of light from a substance that has absorbed light or other electromagnetic radiation. It occurs in certain biological molecules like fireflies, corals, and genetically engineered fish.
2. Fluorescence results from electrons absorbing energy and getting excited to higher energy molecular orbitals, then dropping down and emitting photons of lower energy. The Jablonski diagram illustrates this process.
3. Many factors influence fluorescence, including molecular structure, temperature, solvent, pH, and structural rigidity. Fluorescent dyes like FITC and cyanine dyes are used in applications like labeling and fluorescence resonance energy transfer.
This document discusses optical rotatory dispersion (ORD) and circular dichroism (CD). ORD measures the change in specific rotation of plane-polarized light with wavelength for optically active compounds. CD measures the difference in absorption of left and right circularly polarized light. Key differences are that ORD uses plane-polarized light while CD uses circularly polarized light. ORD graphs plot specific rotation versus wavelength while CD graphs plot molar ellipticity versus wavelength. Both techniques provide information about molecular structure and can be used to analyze proteins, nucleic acids, and other biomolecules.
Infrared spectroscopy involves using infrared radiation to analyze materials. Molecules absorb specific infrared frequencies that are characteristic of their structure, such as bond vibrations and stretches. There are two main methods for infrared spectroscopy - scanning monochromator which analyzes one wavelength at a time, and Fourier transform infrared spectroscopy which uses interferometry to measure all infrared wavelengths simultaneously. Fourier transform then converts this raw interferogram data into the infrared spectrum. Infrared spectroscopy can be used to identify functional groups and molecular structures in compounds like 1-Hexene, Toluene, and Cyclohexanol based on their characteristic absorption peaks.
The all the content in this profile is completed by the teachers, students as well as other health care peoples.
thank you, all the respected peoples, for giving the information to complete this presentation.
this information is free to use by anyone.
Chromatography is a method of separating components of a mixture through their interactions with two phases - a stationary phase and a mobile phase. The components are distributed between the phases based on properties like solubility and affinity. There are several types of chromatography classified by the shape of the stationary phase (e.g. thin layer), the state of the mobile phase (e.g. gas, liquid), or the interaction between solute and stationary phase (e.g. adsorption, partition). Chromatography techniques are used in various applications including pharmaceutical quality control, forensic analysis, and biological research like protein purification.
This document provides an overview of nuclear magnetic resonance (NMR) spectroscopy. It begins with a brief introduction to NMR and its two main types - 1H NMR and 13C NMR. The document then covers the history and development of NMR, including important discoveries and Nobel Prizes. It describes the basic principles and theory of NMR spectroscopy, including nuclear spin, resonance frequency, and chemical shifts. The document discusses NMR instrumentation and experimental aspects such as solvents, spectra, and splitting patterns. It also covers carbon-13 NMR and applications of NMR spectroscopy such as structure elucidation and determination of optical purity.
Flourescence spectroscopy- instrumentation and applicationssinghsnehi01
This document discusses fluorescence and phosphorescence. It defines fluorescence as the emission of light that starts immediately upon absorption of light and stops when the light is removed. Phosphorescence is defined as delayed fluorescence where light continues to be emitted even after the absorbed light is removed. It discusses factors that affect fluorescence like concentration, quantum yield, incident light intensity, oxygen, pH, temperature, viscosity, photodecomposition, and quenchers. Instrumentation for fluorescence includes light sources, filters, sample cells, monochromators, and detectors like photomultiplier tubes. Applications include determination of metals in alloys and fluorescence-based assays.
This document discusses fluorimetry and phosphorimetry. It defines them as measurement techniques, with fluorimetry measuring fluorescence intensity at a particular wavelength, and phosphorimetry measuring phosphorescence in conjunction with pulsed radiation. It describes the principles behind photoluminescence, including fluorescence and phosphorescence. Factors affecting these processes and instrumentation used are summarized, including light sources, filters, monochromators, and detectors. Applications in pharmaceutical, clinical, environmental, and entertainment fields are also briefly outlined.
This document provides an overview of flame emission spectroscopy and atomic absorption spectroscopy. It discusses the theory, principles, instrumentation, interferences and applications of both techniques. Flame emission spectroscopy measures the light emitted from excited atoms in a flame, while atomic absorption spectroscopy measures the absorption of light by ground state atoms. Both can be used to analyze metals but atomic absorption spectroscopy provides better precision and is applicable to more elements.
This document describes continuous wave (CW) NMR spectroscopy. It explains that CW-NMR uses a fixed magnetic field and varies the frequency coil current to achieve resonance signals. A typical CW spectrometer contains a sample tube spun between the poles of a powerful magnet. Radio frequency is broadcast into the sample while a receiver coil monitors the absorbed energy. By varying the magnetic field or radio frequency, an NMR spectrum is acquired showing signals from different proton types in the sample. While offering routine 1H NMR studies, CW spectroscopy has limitations such as low sensitivity, requirement for concentrated samples, and production of high noise.
Flash chromatography is a rapid technique for separating mixtures that uses slightly smaller silica gel particles and pressurized gas to drive the solvent through the column. It is faster than traditional column chromatography while still providing good resolution. Modern systems use pre-packed cartridges and pumps to automate the process. Flash chromatography is well-suited for preparative separations and purification of samples prior to further analysis.
This document discusses several factors that can affect UV-Vis absorption spectra, including sample temperature, concentration, pH, solvent, and molecular structure. Lowering the temperature results in sharper, more defined absorption bands. Increasing the concentration can lead to band broadening at high levels due to molecular interactions. Changing the pH can cause shifts in absorption maxima for compounds like phenols and anilines. The solvent can also impact absorption by stabilizing different electronic states to varying degrees. Molecular structure factors such as conjugation, steric hindrance, and isomerism further influence absorption spectra.
IR SPECTROSCOPY, INTRODUCTION, PRINCIPLE, THEORY, FATE OF ABSORBED RADIATION, FERMI RESONANCE, FINGERPRINT REGION, VIBRATIONS, FACTORS AFFECTING ABSORPTION OF IR RADIATION, SAMPLING TECHNIQUES, APPLICATIONS OF IR SPECTROSCOPY.
In this slide contains instrumentation of Fourier-Transform Nuclear Magnetic Resonance (FT-NMR).
Presented by: P. Venkatesh. (Department of pharmaceutical analysis).
RIPER, anantpur.
A. 13C NMR spectroscopy provides information about carbon structures in organic compounds. It measures the small differences in magnetic field strength needed for carbon nuclei to resonate. These differences are reported in parts per million (ppm) relative to tetramethylsilane (TMS) as a standard. Factors like electronegativity, hybridization, and hydrogen bonding affect the chemical shift values. 13C NMR has applications in metabolic studies and industrial analyses of solids.
spectrofluorometer is the instrument for recording fluorescence emission and absorption spectra 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.
The document provides information about electroanalytical methods of analysis. It defines electroanalytical methods as techniques that study analytes by measuring potentials or currents in an electrochemical cell containing the analyte. It discusses various types of electroanalytical techniques including potentiometry, voltammetry, and Karl Fischer titration. It provides details on the principles, instrumentation, applications, and advantages of these analytical methods.
Fluorometry is an analytical technique that uses fluorescence to identify and characterize small amounts of substances. It involves exciting a sample with ultraviolet or visible light, which causes certain molecules to absorb energy and reach an excited electronic state. The molecules then emit light of a longer wavelength as they fall back to the ground state, and the intensity and composition of this fluorescent light can be measured. Fluorometric methods have applications in pharmaceutical analysis to measure compounds like riboflavin, thiamine, and reserpine in drug formulations.
Fluorimetry, principle, Concept of singlet,doublet,and triplet electronic sta...Vandana Devesh Sharma
This document discusses the principles and factors affecting fluorescence and fluorimetry. It begins by defining fluorescence as the emission of light by a substance that has absorbed light or other electromagnetic radiation. It then discusses various processes that can occur in excited molecules including fluorescence, phosphorescence, internal conversion, intersystem crossing, and collisional deactivation. The document also summarizes several factors that can influence fluorescence intensity, including molecular structure, temperature, viscosity, oxygen content, and pH. Structural factors discussed include conjugation, substituent groups, and molecular rigidity.
This document provides an overview of fluorescence spectroscopy. It describes how luminescence occurs when a system absorbs external energy like light and emits photons. Specifically, fluorescence involves absorbing ultraviolet or visible light which causes molecule excitation, then reemission of light. The document outlines fluorescence instrumentation components like light sources, wavelength selection using filters or monochromators, detectors, and sample holders. It also discusses related topics such as phosphorescence, absorption spectra, and the advantages and disadvantages of fluorescence spectroscopy.
Phosphorescence is the process by which materials emit light after absorbing radiation or energy. It was first observed in the 17th century but not studied scientifically until the 19th century. Phosphorescent materials store absorbed energy and release it slowly as light over minutes or hours by trapping electrons in an excited state. There are two types of photoluminescence - fluorescence which emits light quickly, and phosphorescence which emits light slowly. Common phosphorescent materials include zinc sulfide and strontium aluminate.
1. Fluorescence is the emission of light from a substance that has absorbed light or other electromagnetic radiation. It occurs in certain biological molecules like fireflies, corals, and genetically engineered fish.
2. Fluorescence results from electrons absorbing energy and getting excited to higher energy molecular orbitals, then dropping down and emitting photons of lower energy. The Jablonski diagram illustrates this process.
3. Many factors influence fluorescence, including molecular structure, temperature, solvent, pH, and structural rigidity. Fluorescent dyes like FITC and cyanine dyes are used in applications like labeling and fluorescence resonance energy transfer.
This document discusses optical rotatory dispersion (ORD) and circular dichroism (CD). ORD measures the change in specific rotation of plane-polarized light with wavelength for optically active compounds. CD measures the difference in absorption of left and right circularly polarized light. Key differences are that ORD uses plane-polarized light while CD uses circularly polarized light. ORD graphs plot specific rotation versus wavelength while CD graphs plot molar ellipticity versus wavelength. Both techniques provide information about molecular structure and can be used to analyze proteins, nucleic acids, and other biomolecules.
Infrared spectroscopy involves using infrared radiation to analyze materials. Molecules absorb specific infrared frequencies that are characteristic of their structure, such as bond vibrations and stretches. There are two main methods for infrared spectroscopy - scanning monochromator which analyzes one wavelength at a time, and Fourier transform infrared spectroscopy which uses interferometry to measure all infrared wavelengths simultaneously. Fourier transform then converts this raw interferogram data into the infrared spectrum. Infrared spectroscopy can be used to identify functional groups and molecular structures in compounds like 1-Hexene, Toluene, and Cyclohexanol based on their characteristic absorption peaks.
The all the content in this profile is completed by the teachers, students as well as other health care peoples.
thank you, all the respected peoples, for giving the information to complete this presentation.
this information is free to use by anyone.
Chromatography is a method of separating components of a mixture through their interactions with two phases - a stationary phase and a mobile phase. The components are distributed between the phases based on properties like solubility and affinity. There are several types of chromatography classified by the shape of the stationary phase (e.g. thin layer), the state of the mobile phase (e.g. gas, liquid), or the interaction between solute and stationary phase (e.g. adsorption, partition). Chromatography techniques are used in various applications including pharmaceutical quality control, forensic analysis, and biological research like protein purification.
This document provides an overview of nuclear magnetic resonance (NMR) spectroscopy. It begins with a brief introduction to NMR and its two main types - 1H NMR and 13C NMR. The document then covers the history and development of NMR, including important discoveries and Nobel Prizes. It describes the basic principles and theory of NMR spectroscopy, including nuclear spin, resonance frequency, and chemical shifts. The document discusses NMR instrumentation and experimental aspects such as solvents, spectra, and splitting patterns. It also covers carbon-13 NMR and applications of NMR spectroscopy such as structure elucidation and determination of optical purity.
Flourescence spectroscopy- instrumentation and applicationssinghsnehi01
This document discusses fluorescence and phosphorescence. It defines fluorescence as the emission of light that starts immediately upon absorption of light and stops when the light is removed. Phosphorescence is defined as delayed fluorescence where light continues to be emitted even after the absorbed light is removed. It discusses factors that affect fluorescence like concentration, quantum yield, incident light intensity, oxygen, pH, temperature, viscosity, photodecomposition, and quenchers. Instrumentation for fluorescence includes light sources, filters, sample cells, monochromators, and detectors like photomultiplier tubes. Applications include determination of metals in alloys and fluorescence-based assays.
This document discusses fluorimetry and phosphorimetry. It defines them as measurement techniques, with fluorimetry measuring fluorescence intensity at a particular wavelength, and phosphorimetry measuring phosphorescence in conjunction with pulsed radiation. It describes the principles behind photoluminescence, including fluorescence and phosphorescence. Factors affecting these processes and instrumentation used are summarized, including light sources, filters, monochromators, and detectors. Applications in pharmaceutical, clinical, environmental, and entertainment fields are also briefly outlined.
This document provides an overview of flame emission spectroscopy and atomic absorption spectroscopy. It discusses the theory, principles, instrumentation, interferences and applications of both techniques. Flame emission spectroscopy measures the light emitted from excited atoms in a flame, while atomic absorption spectroscopy measures the absorption of light by ground state atoms. Both can be used to analyze metals but atomic absorption spectroscopy provides better precision and is applicable to more elements.
This document describes continuous wave (CW) NMR spectroscopy. It explains that CW-NMR uses a fixed magnetic field and varies the frequency coil current to achieve resonance signals. A typical CW spectrometer contains a sample tube spun between the poles of a powerful magnet. Radio frequency is broadcast into the sample while a receiver coil monitors the absorbed energy. By varying the magnetic field or radio frequency, an NMR spectrum is acquired showing signals from different proton types in the sample. While offering routine 1H NMR studies, CW spectroscopy has limitations such as low sensitivity, requirement for concentrated samples, and production of high noise.
Flash chromatography is a rapid technique for separating mixtures that uses slightly smaller silica gel particles and pressurized gas to drive the solvent through the column. It is faster than traditional column chromatography while still providing good resolution. Modern systems use pre-packed cartridges and pumps to automate the process. Flash chromatography is well-suited for preparative separations and purification of samples prior to further analysis.
This document discusses several factors that can affect UV-Vis absorption spectra, including sample temperature, concentration, pH, solvent, and molecular structure. Lowering the temperature results in sharper, more defined absorption bands. Increasing the concentration can lead to band broadening at high levels due to molecular interactions. Changing the pH can cause shifts in absorption maxima for compounds like phenols and anilines. The solvent can also impact absorption by stabilizing different electronic states to varying degrees. Molecular structure factors such as conjugation, steric hindrance, and isomerism further influence absorption spectra.
IR SPECTROSCOPY, INTRODUCTION, PRINCIPLE, THEORY, FATE OF ABSORBED RADIATION, FERMI RESONANCE, FINGERPRINT REGION, VIBRATIONS, FACTORS AFFECTING ABSORPTION OF IR RADIATION, SAMPLING TECHNIQUES, APPLICATIONS OF IR SPECTROSCOPY.
In this slide contains instrumentation of Fourier-Transform Nuclear Magnetic Resonance (FT-NMR).
Presented by: P. Venkatesh. (Department of pharmaceutical analysis).
RIPER, anantpur.
A. 13C NMR spectroscopy provides information about carbon structures in organic compounds. It measures the small differences in magnetic field strength needed for carbon nuclei to resonate. These differences are reported in parts per million (ppm) relative to tetramethylsilane (TMS) as a standard. Factors like electronegativity, hybridization, and hydrogen bonding affect the chemical shift values. 13C NMR has applications in metabolic studies and industrial analyses of solids.
spectrofluorometer is the instrument for recording fluorescence emission and absorption spectra 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.
The document provides information about electroanalytical methods of analysis. It defines electroanalytical methods as techniques that study analytes by measuring potentials or currents in an electrochemical cell containing the analyte. It discusses various types of electroanalytical techniques including potentiometry, voltammetry, and Karl Fischer titration. It provides details on the principles, instrumentation, applications, and advantages of these analytical methods.
Fluorometry is an analytical technique that uses fluorescence to identify and characterize small amounts of substances. It involves exciting a sample with ultraviolet or visible light, which causes certain molecules to absorb energy and reach an excited electronic state. The molecules then emit light of a longer wavelength as they fall back to the ground state, and the intensity and composition of this fluorescent light can be measured. Fluorometric methods have applications in pharmaceutical analysis to measure compounds like riboflavin, thiamine, and reserpine in drug formulations.
Fluorimetry, principle, Concept of singlet,doublet,and triplet electronic sta...Vandana Devesh Sharma
This document discusses the principles and factors affecting fluorescence and fluorimetry. It begins by defining fluorescence as the emission of light by a substance that has absorbed light or other electromagnetic radiation. It then discusses various processes that can occur in excited molecules including fluorescence, phosphorescence, internal conversion, intersystem crossing, and collisional deactivation. The document also summarizes several factors that can influence fluorescence intensity, including molecular structure, temperature, viscosity, oxygen content, and pH. Structural factors discussed include conjugation, substituent groups, and molecular rigidity.
Introduction, theoretical principle, quantum efficiency of fluorescence, molecular structure of
fluorescence, instrumentation, factors influencing the intensity of fluorescence, comparison of
fluorometry with spectrophotometry, application of fluorometry in pharmaceutical analysis
1. Fluorescence spectrophotometry measures the intensity of light emitted by a substance that has absorbed ultraviolet or visible light.
2. After light absorption, molecules can deactivate through radiationless processes like internal conversion or intersystem crossing, or through emission of a photon during fluorescence or phosphorescence.
3. Factors like a molecule's structure, solvent, temperature, and pH can affect its fluorescence quantum yield by changing rates of radiationless relaxation versus light emission.
This document is a student's report on luminescence spectroscopy submitted to their professor. It defines fluorescence and phosphorescence, explaining the principles using the Jablonski diagram. Fluorescence occurs from the first excited singlet state and involves emission of a photon within nanoseconds of absorbing light. Phosphorescence involves intersystem crossing to the triplet state, with emission of a photon over micro- to milliseconds. The key differences are that fluorescence stops immediately upon removing excitation, while phosphorescence emission persists afterwards due to the longer-lived triplet state.
Fluorimetry.pptx by Saloni Kadam Nanded talukauser621767
The document discusses fluorimetry and provides details about:
- Luminescence processes including fluorescence and phosphorescence
- Factors that affect fluorescence like pH, temperature, and concentration
- Instrumentation used for fluorimetry including radiation sources, monochromators, sample holders, and photomultiplier tube detectors
- Quenching processes that can decrease fluorescence intensity
The document discusses the theory of fluorimetry. It begins by defining luminescence as light emission from a substance when an electron returns to the ground state from an excited state. It then describes the three types of luminescence - fluorescence, phosphorescence, and chemiluminescence. Fluorescence occurs immediately when light is absorbed, while phosphorescence occurs more slowly after light is removed. Fluorimetry is the measurement of fluorescence, involving excitation and emission spectra. The document goes on to discuss singlet and triplet electronic states, Stokes shift, lifetime, quantum yield, and references in the field of fluorimetry.
Fluorescence is a type of luminescence where molecules emit light from electronically excited states created by light absorption. The fluorescence process involves three steps: 1) excitation of a molecule to an excited electronic state, 2) vibrational relaxation to the lowest excited vibrational level, and 3) emission of a photon and return to the ground state. Phosphorescence also involves light emission from an excited state, but occurs from a longer-lived triplet excited state following intersystem crossing. The absorption and fluorescence emission spectra of molecules generally overlap but the fluorescence peaks are at slightly longer wavelengths due to a Stokes shift.
1. Fluorimetry is the measurement of fluorescence intensity using a fluorimeter or spectrofluorimeter. It involves exciting a molecule with specific wavelengths of light which causes electrons to get promoted to excited states, then relax emitting light of longer wavelengths.
2. Key components of fluorimeters include light sources like mercury lamps, filters to select wavelengths, sample cells, and detectors like photomultiplier tubes. Spectrofluorimeters use monochromators instead of filters to isolate wavelengths.
3. Fluorescence can be quenched by factors like oxygen, pH, temperature. Applications include determining inorganic/organic substances, proteins, pigments in nanogram amounts.
Exp 5: Chemiluminescence Name: Date: Post-Lab Questions: 1. Identify a common structural feature of the fluorophores used in this experiment. (2pts) 2. Explain the difference between fluorescence and phosphorescence. (4 pts)
Solution
1. All these fluorophores are aromatic . These substances absorbs energy from the source of light and shows electronic transitions from ground to excited state and this excited electron looses energy in form of radiation leading to light phenomenon .
2\'. Fluorescence is the phenomenon in which excited electron loose energy in form of light radiations while coming from first singlet excited state to ground singlet state . It of short time phenomenon. That is S^1 = S^0 + light
While phosphorescence is phenomenon of loosening of energy in form of light radiations from excited e- from first triplet excited state to singlet ground state .
That is T^1 = S^0 + light .
As transition from triplet state to singlet state is spin forbidden , so this is longer time phenomenon as compare to fluorescence.
.
3.2 molecular fluorescence and phosphorescence spectroscopyGaneshBhagure2
This document discusses molecular fluorescence and phosphorescence spectroscopy. It begins with an introduction to the principles and terms, explaining that fluorescence occurs when emission takes place within 10-8 seconds of absorption, while phosphorescence occurs after more than 10-8 seconds. The document then covers electronic transitions, factors that affect fluorescence and phosphorescence like temperature, pH, and solvent, and instrumentation including components of fluorimeters.
What is Fluorescence Electrons in an atom or a m.pdfapnashop1
What is Fluorescence? Electrons in an atom or a molecule can absorb the energy in
the electromagnetic radiation and thereby excite to an upper energy state. This upper energy state
is unstable; therefore, electron likes to come back to the ground state. When coming back, it
emits the absorbed wavelength. In this relaxation process, they emit excess energy as photons.
This relaxation process is known as fluorescence. Fluorescence takes place much more rapidly.
Generally, it completes in about 10-5 s or less time from the time of excitation. In atomic
fluorescence, gaseous atoms fluoresce when they are exposed to radiation with a wavelength that
exactly matches one of the absorption lines of the element. For example, gaseous sodium atoms
absorb and excite by absorbing 589 nm radiations. Relaxation takes place after this by
reemission of fluorescent radiation of the identical wavelength. Because of this, we can use
fluorescence to identify different elements. When excitation and reemission wavelengths are the
same, the resulting emission is called resonance fluorescence. Other than fluorescence, there are
other mechanisms by which an excited atom or molecule can give up its excess energy and relax
to its ground state. Nonradiative relaxation and fluorescence emissions are two such important
mechanisms. Because of many mechanisms, the lifetime of an excited state is brief. The relative
number of molecules that fluoresce is small because fluorescence requires structural features that
slow the rate of the nonradiative relaxation and enhance the rate of fluorescence. In most
molecules, these features are not there; therefore, they undergo nonradiative relaxation, and
fluorescence does not occur. Molecular fluorescence bands are made up of a large number of
closely spaced lines; therefore, usually it is hard to resolve. What is Phosphorescence? When
molecules absorb light and go to the excited state they have two options. They can either release
energy and come back to the ground state immediately or undergo other non-radiative processes.
If the excited molecule undergoes a non radiative process, it emits some energy and come to a
triplet state where the energy is somewhat lesser than the energy of the exited state, but it is
higher than the ground state energy. Molecules can stay a bit longer in this less energy triplet
state. This state is known as the metastable state. Then metastable state (triplet state) can slowly
decay by emitting photons, and come back to the ground state (singlet state). When this happens
it is known as phosphorescence. What is the difference between Fluorescence and
Phosphorescence? • When light is supplied to a sample of molecules, we immediately see the
fluorescence. Fluorescence stops as soon as we take away the light source. But phosphorescence
tends to stay little longer even after the irradiating light source is removed. • Fluorescence takes
place when excited energy is released, and the molecule comes back to the gro.
This document discusses molecular fluorescence and phosphorescence spectroscopy. It provides details on:
- The processes of fluorescence, where emission occurs from an excited singlet state, and phosphorescence, where emission occurs from an excited triplet state.
- Factors that influence fluorescence and phosphorescence intensities like quantum yield, transition types (π->π* vs π->n), molecular structure, and competing deactivation processes.
- How excitation and emission spectra are obtained and related to the energy of electronic state transitions.
- Applications of photoluminescence spectroscopy like determining material band gaps and quality.
This chapter discusses fluorometry, which uses fluorescence to perform sensitive analyses. Fluorescence occurs when molecules absorb ultraviolet or visible light and emit light of a lower energy as they return to the ground state. Factors that influence fluorescence intensity include concentration, presence of other solutes, pH, temperature, and chemical structure. Fluorometry is compared to spectrophotometry, with fluorometry typically being more sensitive but less specific. Applications of fluorometry to pharmaceutical analysis are discussed, particularly for analyzing drugs and metabolites in biological samples.
1. Photochemistry involves using light as a chemical reagent to promote molecules to electronically excited states or as a chemical product when excited states return to the ground state.
2. Fluorescence occurs when a molecule in an excited singlet state returns to the ground state and emits light. It is a spin-allowed process that results in emission at a longer wavelength than the absorbed light.
3. The excitation and fluorescence emission spectra of a compound are often approximately mirror images of each other, though there are exceptions when the excited and ground states differ in geometry.
This document presents an overview of fluorimetry. It discusses that fluorimetry is the measurement of emitted fluorescence light. When certain substances are exposed to light, they emit visible light or radiation, known as fluorescence. Fluorescence occurs immediately after light absorption and stops when the light is removed. Phosphorescence is delayed fluorescence that continues after light removal. Fluorimetry works by exciting substances from their singlet ground state to a singlet excited state, then measuring the wavelength of light emitted as they return to the ground state.
Fluorescence spectroscopy involves exciting a chemical substance with UV or visible light, causing it to emit light at a longer wavelength. This phenomenon is called luminescence. Depending on the lifetime of the excited state, luminescence is categorized into fluorescence (10-8 to 10-4 seconds) or phosphorescence (10-4 to 10 seconds). Fluorometry is a sensitive analytical technique used in various fields like biochemistry and environmental science to study molecular interactions and assay fluorescent compounds like drugs. It provides high selectivity for trace analysis of substances in biological samples.
This document discusses fluorescence and phosphorescence techniques for chemical analysis. It defines fluorescence as emission of light that occurs immediately when excited by light, while phosphorescence involves continuous light emission even after excitation stops. Factors that determine whether a molecule is fluorescent include its quantum yield, structure, temperature, solvent, concentration, and pH. Instrumentation for fluorimetry includes light sources, filters, monochromators, and photomultiplier tubes. Applications include determination of inorganic and organic species. Fluorimetry offers higher sensitivity, specificity, and precision compared to absorption spectroscopy.
Fluorescence and phosphorescence are forms of luminescence that involve the emission of light from a substance that has absorbed radiation or light. Fluorescence involves emission of light from singlet excited states, while phosphorescence involves emission from triplet excited states. Factors like temperature, concentration, and molecular structure can influence the intensity of fluorescence. Fluorescence and phosphorescence find applications in areas like analytical chemistry, microscopy, lighting, and more. Instrumentation used to study these phenomena include filter fluorimeters and modern fluorescence spectrophotometers.
The document discusses photoluminescence, which is the emission of light from a material when it absorbs photons. There are three main steps in the photoluminescence process: excitation, relaxation, and emission. Excitation occurs when photons are absorbed and electrons are lifted to a higher energy state. Relaxation follows as electrons lose energy non-radiatively. Emission is the radiative decay of electrons as they return to the ground state, emitting photons of lower energy than those absorbed. The two main types of photoluminescence are fluorescence, which is a rapid emission, and phosphorescence, which is a slower emission.
Vitamins & vitamin containing drugs manikImran Nur Manik
Vitamins are organic compounds that are essential nutrients for the human body. There are 13 essential vitamins that must be obtained through diet as the body cannot synthesize them. Vitamins play important roles in growth, development, and metabolic processes. Deficiencies can lead to specific diseases. Vitamins can be fat-soluble like A, D, E and K which are stored in the body, or water-soluble like the B vitamins and C which are not stored. Dietary sources and functions of several key vitamins are discussed.
Standardization of Acids and bases.
2. Determination of pKa and pKb values
3. Preparation of solutions of different pH & buffer capacities.
4. Determination of phase diagram of binary systems.
Determination of distribution coefficients.
6. Determination of molecular weight by Victor Meyer’s Method.
7. Determination of heats of solutions by measuring solubility as a function of temperature
(Van’t Hoff equation.)
A. Qualitative analysis of metal ions and acid radicals:
Na+, K+, Ca+2, Ag+, Mn+4, Fe+2, Fe+3, Co+2, Mg+2, Al+3, Cu+2 and acid radicals CO3,
halides, Citrate
SO4-2, NO3-, SO3-2, etc.
B. Identification of inorganic drugs in their formulation:
1. Ca+2, from supplied preparations
2. Fe+2 from supplied preparations
3. Al+3 from supplied preparations
4. Mg+2 from supplied preparations
5. K+ from supplied reparations
6. Na+ from supplied preparations
C. Conversion of different water insoluble or sparingly soluble drugs into water soluble
forms:
1. Na/ K – salicylate from salicylic acid
2. Na/ K – benzoate from benzoic acid
3. Na/ K – citrate from citric acid
Plants in complimentary and traditional systems of medicine MANIKanikImran Nur Manik
Plants in complimentary and traditional systems of medicine: Introduction-different types of
alternative systems of treatments (e.g. Ayurvedic, Unani and Homeopathic medicine). Contribution
of traditional drugs to modern medicines. Details of some common indigenous traditional drugs:
Punarnava, Vashaka, Anantamul, Arjuna, Chirata, Picrorhiga, Kalomegh, Amla, Asoka, Bahera,
Haritaki, Tulsi, Neem, Betel nut, Joan, Karela, Shajna, Carrot, Bael, Garlic, Jam and Madar.
This document provides information about various lipids (fats and oils) obtained from plants and animals. It discusses the basic chemistry of lipids, describing them as esters of fatty acids and alcohols. Specific lipids are then outlined, including their source, composition, properties, and some uses. Key lipids discussed include olive oil, coconut oil, castor oil, linseed oil, peanut oil, chaulmoogra oil, and beeswax.
Pharmacognosy is the study of medicinal plants and natural products. The term was introduced in 1815 and comes from Greek roots meaning "drug" and "knowledge." It involves the study of plants as potential drug sources from pre-historic use through various civilizations like Chinese, Babylonian, Egyptian, Indian, and Greek. Modern pharmacognosy has broad applications in medicine, agriculture, cosmetics, and other industries and offers career opportunities in academia, private industry, and government.
Crude drugs: A general view of their origin, distributions, cultivation, collection, drying and
storage, commerce and quality control.
a) Classification of drugs.
b) Preparation of drugs for commercial market
c) Evaluation of crude drugs.
d) Drug adulteration.
Carbohydrates are organic compounds made of carbon, hydrogen, and oxygen. They play a vital role in life and include monosaccharides (simple sugars), disaccharides, and polysaccharides. Common monosaccharides are glucose and fructose. Sucrose is a prevalent disaccharide composed of glucose and fructose. Starch and cellulose are examples of polysaccharides. Carbohydrates serve important functions and some like glucose are used as nutrients. Tests can identify the presence of carbohydrates and their type.
The document discusses alkaloids, which are nitrogen-containing plant compounds. It defines alkaloids and explains that they are difficult to define precisely due to overlapping properties with other amines. It then covers the distribution of various alkaloids in different plant parts, their chemical properties, pharmacological actions, classification based on ring structure, extraction methods, and chemical tests to identify alkaloids.
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Students should calculate the time allotted per mark on their exam to help manage their time efficiently. For example, a 40 mark exam in 2.25 hours means each mark is worth 3 minutes and 22 seconds. Students should also practice solving previous years' exam questions and ensure they have the proper stationaries like pens, pencils, erasers and papers like admit cards for their exam. Proper preparation of time management and materials can help students complete their written exams successfully.
Volatile oils and related terpenoids-Methods of obtaining volatile oils,
chemistry, their medicinal and commercial uses, biosynthesis of some important
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1. Describe the organisation of respiratory center
2. Describe the nervous control of inspiration and respiratory rhythm
3. Describe the functions of the dorsal and respiratory groups of neurons
4. Describe the influences of the Pneumotaxic and Apneustic centers
5. Explain the role of Hering-Breur inflation reflex in regulation of inspiration
6. Explain the role of central chemoreceptors in regulation of respiration
7. Explain the role of peripheral chemoreceptors in regulation of respiration
8. Explain the regulation of respiration during exercise
9. Integrate the respiratory regulatory mechanisms
10. Describe the Cheyne-Stokes breathing
Study Resources:
1. Chapter 42, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 36, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 13, Human Physiology by Lauralee Sherwood, 9th edition
Promoting Wellbeing - Applied Social Psychology - Psychology SuperNotesPsychoTech Services
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Travel vaccination in Manchester offers comprehensive immunization services for individuals planning international trips. Expert healthcare providers administer vaccines tailored to your destination, ensuring you stay protected against various diseases. Conveniently located clinics and flexible appointment options make it easy to get the necessary shots before your journey. Stay healthy and travel with confidence by getting vaccinated in Manchester. Visit us: www.nxhealthcare.co.uk
8 Surprising Reasons To Meditate 40 Minutes A Day That Can Change Your Life.pptxHolistified Wellness
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Osteoporosis - Definition , Evaluation and Management .pdf
Fluorometry Manik
1. PHARM 3235
Md. Imran Nur Manik
Lecturer
Department of Pharmacy
Northern University Bangladesh
2. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 1
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Fluorometry
Terminologies
Luminescence: Luminescence is the phenomenon of a chemical species to absorb radiation of UV or
visible region and emit a radiation of longer wavelength. Loss of energy and concomitant transition of
molecules from excited states to ground states with emission of radiation is called luminescence.
What happens here is, energy excites the molecules (more specifically electrons of the molecules). When
the molecules return to the normal state, they emit radiation–light.
Luminescence can be divided into two types depending on the lifespan of the excited state –
1. Fluorescence
2. Phosphorescence
Fluorescence: Fluorescence is defined as the emission of radiation by a chemical species during its
transition from an excited singlet state to the ground (singlet) state. The extent of fluorescence can be
measured by fluorometry.
In the ground state of a molecule, the two electrons responsible for bonding lie in
the bonding molecular orbital in opposite spins. Now when energy is applied to
excite the molecule, one of the electrons will transit to the excited state i.e. the
antibonding molecular orbital. If the excited electron in the antibonding orbital has
spin opposite to the electron present in the bonding orbital of ground sate, then the
excited state is called Excited singlet state.
Ground state Ground state
Excited state
Energy
What is excited singlet state?
At room temperature, in normal condition, molecules will be at ground state and bonding
electrons will spin in opposite directions. Energy level is lowest. Excitation requires energy
which must be supplied in the form of UV or visible light. Following light absorption, a
chromophore is excited to some higher vibrational energy level of S1 or S2. Then, due to
vibrational relaxation, the molecules will descend to the lowest vibrational energy level of the
excited state. This process is radiationless but energy is lost in other forms.
From the lowest vibrational level of excited state, molecules will return to the ground state by
emission of radiation. Since, little energy is lost during vibrational relaxation, the radiation
emitted has lower energy than the radiation absorbed. Hence, in fluorescence emitted radiation
has longer wavelength.
In fluorescent molecules, luminescence stops within 10-8
to 10-4
seconds. It is important to
remember that, the molecule will undergo vibrational relaxation to the lowest vibrational
energy level before returning to the ground state to give fluorescence. So
Md.
Imran
Nur
Manik
3. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 2
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Excited state
Ground state
Energy
Green arrows show
vibrational relaxation
Red arrows indicate
fluorescence
The phenomenon of radiation emission during transition from the lowest vibrational energy
level of the excited singlet state to the ground state is called fluorescence.
Phosphorescence: Phosphorescence is defined as the emission of radiation by a chemical species
during its transition from the excited triplet state to the singlet ground state.
The triplet state of a molecule has a lower energy than its associated singlet state so that
transitions back to the ground state are accompanied with the emission of light of lower energy
than from the singlet state. Therefore, we would typically expect phosphorescence to occur at
longer wavelengths than fluorescence. Phosphorescence is often characterized by an afterglow
because of the long life of the triplet state,10-4-10 seconds.
An important feature of phosphorescence is afterglow. Light is emitted from phosphorescent
molecules after radiation energy source is removed. This is because the luminescence
continues for 10-4
seconds to 10 seconds as the triplet state has greater longevity.
In phosphorescence, similar to the fluorescence, vibrational relaxation must occur.
Excited singlet
state
Ground state
Energy
Green arrows show
vibrational relaxation
Red arrows indicate
phosphorescence
Excited triplet
state
Singlet state: Singlet state is the state in which all of the electrons are paired and in each pair the two
electrons spin about their own axis in opposite directions.
Excited singlet state: When two electrons of the singlet state are goes to the excited state it is called
excited singlet state. In excited singlet state electrons remain as in exciting position.
Md.
Imran
Nur
Manik
4. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 3
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Triplet state: Triplet state is a state lying at an energy level intermediate between ground and excited
state and characterized by an impairing of two electrons.
In contrast to the singlet state, there is a spin reversal involving one electron of the pair and the pair of
two electrons spins about their axis in the same direction. The life time of the molecule in the triplet
state in 10-4 to 10 seconds.
What is excited triplet state?
In the excited singlet state of a molecule, the electron in the excited state
and the electron the ground state spin in opposite direction i.e. they are
still paired. In some compounds, the molecule may convert from the lowest
vibrational level of excited state to a triplet state. In the triplet state, the
electron in excited state spins in the same direction as the electron in the
ground state.
Ground state Ground state
Excited state
Energy Triplet state
Basically the triplet state is the excited state between the ground state and
the excited singlet state and electron in this state spins in the same direction
as that of ground state.
Vibrational energy level:
Even at ground state a molecule is always vibrating. Therefore the energy at ground state is not a single
discreet value rather a set of discreet values. In another words there are different energy levels in the
molecule duo to its vibration.
Whether the molecule is in ground state or excited state, the molecule contains many energy levels
which are called vibrational energy levels.
Excited state
Ground state
Energy
There may be many Occupied Molecular Orbitals (bonding orbitals) and many Unoccupied
Molecular Orbitals (antibonding orbitals-electrons transit to these during excitation). Now,
when a molecule is in ground state, the electrons will not transit to the antibonding orbitals. But
they may transfer from one bonding orbital to another. This causes vibration of the molecules.
Again, when a molecule is in excited state, one electron from each pair of bonding electrons will
transit to the antibonding orbitals. When they are in these antibonding orbital, they may transfer
from one antibonding orbital to another.
Md.
Imran
Nur
Manik
5. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 4
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Vibrational relaxation: Vibrational relaxation is the transition of molecule from any of the vibrational
energy levels to the lowest vibrational energy level of the excitatory state.
Excited state
Ground state
Energy
Green arrows show
vibrational relaxation
Energy lost in this process is thought to be via thermal process probably lost to solvent molecules.
Resonance fluorescence: Resonance fluorescence is the phenomenon where the molecule absorbs
and emits equal amount of energy. Practically resonance fluorescence doesn’t occur or occur rarely as
vibrational relaxation occurs.
Excited state
Ground state
Energy
Green arrows show
absorbed light
Red arrows show
emitted light
Internal conversion: The phenomenon of excited molecule to return to the ground state by losing
energy by means other than photo radiation is termed internal conversion.
Intersystem crossing: The transfer of a molecule present in the lowest vibrational energy level of the
excited singlet state to an excited triplet state is called intersystem crossing.
Differences between fluorescence and phosphorescence:
Property Fluorescence Phosphorescence
Transition Molecule transits from excited singlet state
to ground state.
Molecule transits from excited triplet state to
ground state.
Lifespan Fluorescence is continued for only 10-8 to
10-4 seconds.
Phosphorescence continues for 10-4 seconds
to 10 seconds.
Afterglow Not present. Occurs and luminescence slowly fades.
Analytical
application
Yes. No.
Quantum efficiency: Quantum efficiency is defined as the ratio of number of light quanta emitted and
the number of light quanta absorbed.
absorbedlightofEnergy
emittedlightofEnergy
absorbedquantalightofNo.
emittedquantalightofNo.
orQ
Its significance is that, it is an indicator of how fluorescent a molecule is. If Q is near 1, the molecule is
highly fluorescent molecule and if Q is near 0, the molecule is a very low fluorescent molecule.
Md.
Imran
Nur
Manik
6. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 5
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Fluorometry
The method of analysing a sample by measuring its fluorescence i.e. intensity and composition of light
emitted by it, is called fluorometry.
Fluorescence spectroscopy aka fluorometry or spectrofluorometry is an analytical technique for
identifying and characterizing minute amounts of a fluorescent substance by excitation of the substance
with a beam of ultraviolet light and detection & measurement of the characteristic wavelength of the
fluorescent light emitted.
It is a spectrochemical method. These terms are explained with the illustration below –
Md.
Imran
Nur
Manik
7. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 6
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Excited singlet state
Excited triplet state
Ground state
Blue lines: Vibrational energy level within a state
Green lines: Vibrational relaxation
Sky blue line: Intersysten crossing
Purple lines: Internal conversion (radiationless)
Orange lines: Resonance fluorescence
Red line: Phosphorescence
Black line: Fluorescence
Theory of fluorometry
When energy is applied to certain molecules in the form of UV or visible electromagnetic radiation, the
molecules temporally transit to an excited singlet state where the excited electron is in paired condition
with the ground electron. In the excited state, the molecules lose energy in radiationless manner to
descend to the lowest vibrational energy level of the excited state. The excited state lasts only 10-8 to
10-4 seconds and then the excited molecule will return to ground state by losing energy through
emitting radiation. This is termed fluorescence and the emitted radiation is of longer wavelength.
By measuring the emitted wavelength we can determine the presence and amount of a compound in a
sample.
Md.
Imran
Nur
Manik
8. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 7
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
1. When a molecule absorbs radiant energy it is got promoted from the ground state to the excited state
and gets distributed in the various vibrational energy levels mostly to the excited singlet state.
2. Radiationless vibrational relaxation to the lowest vibrational energy level of the excited singlet state:
Molecules initially undergo a more rapid process, a radiationless loss of vibrational energy and so
quickly falls to the lowest vibrational energy level of the excited state, known as vibrational relaxation.
3. Radiationless internal conversion (from excited singlet state to ground state followed by vibrational
relaxation): From the lowest vibrational energy level of the excited singlet state, a molecule can return to
the ground state by photoemission or by radiationless process followed by vibrational relaxation.
When an excited molecule undergo a radiationless loss of vibrational energy, sufficient to drop to the ground state
then it is termed internal conversion.
4. Fluorescence (Followed by vibrational relaxation):The radiation emitted in the transition of a molecule
from a singlet excited state to a singlet ground state is called fluorescence.
The radiation emitted as fluorescence is of lower energy and therefore of longer wavelength than that originally
absorbed.
5. Intersystem crossing (From excited singlet state to excited triplet state): Molecule in the lowest
vibrational energy level of the excited singlet state converts to a triplet state (the state lying at an energy
level intermediate between ground state and excited).This process is called intersystem crossing. Here
molecules do not losses energy.
6. Vibrational relaxation (to the lowest vibrational energy level of the excited triplet state): Once
intersystem crossing has occurred, a molecule so quickly falls to the lowest vibrational energy level of
the excited triplet state by vibrational relaxation. The lifetime of molecule in the triplet state is 10-4 to
10 seconds (Longer than corresponding singlet state).
Md.
Imran
Nur
Manik
9. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 8
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
7. Radiationless internal conversion from excited triplet state to ground state followed by vibrational
relaxation: Here energy is released in the form of heat radiation.
8. Phosphorescence (Followed by vibrational relaxation). The emission of radiation emitted in the
transition of a molecule from a triplet excited state to a singlet ground state is called Phosphorescence.
It is characterized by afterglow because of the long life of the triplet state.
Relationship between fluorescence and chemical structure
Definite correlations between chemical structure and fluorescence can’t be made. But there is influence
of structural features on the fluorescence of organic compounds.
Degree of conjugation: Conjugation is necessary for fluorescence. This is because mobile π electrons are
responsible for UV-Vis absorption characteristics of compounds. Thus cyclohexane (saturated, no π
electron) is not fluorescent, benzene is weakly fluorescent and anthracene is highly fluorescent.
Napthalene
(strongly fluorescent)
Benzene
(weakly
fluorescent)
Cyclohexane
(non-fluorescent)
Delocalization of electron: In mono-substituted benzene derivatives following rules can apply –
Methyl and other alkyl groups have little effect on fluorescence intensity.
Electron donating groups i.e. ortho-para directors increase fluorescence intensity as they
increase electron density (increase electron delocalization). E.g. fluoro, amino, hydroxy, methoxy
group etc.
Exception: Halogen substitution specifically chlorine, bromine and iodine substitution
decreases/diminishes fluorescence by causing intersystem crossing. Thus they show
phosphorescence.
Electron withdrawing groups i.e. meta directors decrease fluorescence intensity as they decrease
electron density (causes π electron localization). E.g. carboxyl, nitro, sulfonyl, aldehyde group etc.
Exception: Nitrile group even though meta directing, increases fluorescence intensity.
It was postulated that electrons of the CN group interacted with the π electrons of the benzene ring to result in a
distribution that favoured fluorescence.
In di-substituted benzene derivatives fluorescence is
unpredictable. For example, aniline is a fluorescent
compound. When a meta directing group e.g.
sulfamoyl group is added (then the compound is
sulfanilamide) fluorescent intensity increases 5 times.
Although it may be expected that substitution of the fluorescent compound, aniline, with a meta-directing group —
SO2NH2 would result in a compound which would fluoresce to a lesser degree than aniline.But Sulfanilamide, however,
was found to be five times as fluorescent as aniline.
Compound Fluorescence compare to benzene
Higher Lower
Benzaldehyde
Chlorobenzene
Aniline
Nitrobenzene
Benzoic acid
Phenol
Md.
Imran
Nur
Manik
10. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 9
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Molecular geometry:
Rigidity and planarity: The higher the rigidity the greater is the fluorescence intensity. This is because,
rigidity and planarity will prevent vibration and free rotation of aromatic rings hence less energy is
dissipated in radiationless manner.
(Fluorescein , is highly fluorescent, while phenolphthalein is nonfluorescent. The oxygen bridge in fluorescein imparts rigidity and
planarity that is not present in phenolphthalein. the vibrational energy is greater.)
OO O
COO
O
COO
O
Fluorescin
(strongly fluorescent)
Phenolphthalein
(non-fluorescent)
cis-trans isomerism: It also affects fluorescence intensity. Generally trans isomers have greater
fluorescence than corresponding cis isomers. This is due to non-planar character of cis isomers.
CH
HC
HC
HC
cis-stilbenetrans-stilbene
Heterocylic compounds:
N
H
O S
Decreased fluorescence
intensity
Pyrrole
N
2H-PyrroleFuran Thiophene
Increased fluorescence intensity
A double-bonded nitrogen (=N—) generally decrease the fluorescence intensity but S,O,NH
generally increase the fluorescence intensity.
Ionization: Many compounds show fluorescence at ionized state. But this is dependent upon pH of the
solution.
Complexation: Complexation increases rigidity and minimizes internal vibration hence fluorescence
intensity is increased.
e.g. Tetracycline has a weak native fluorescence but complexes of the antibiotic with Ca2+ and a
barbiturate fluorescence quiet intensely.
Tetracycline→ complexes
(non fluorescent) (fluorescent)
Md.
Imran
Nur
Manik
11. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 10
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Chemical conversion
Acid treatment-
Hydrocortisone is not fluorescent itself but they from strongly fluorescence compound in concentrated
in the prescence of ethanol.
Hydrocortisone → strongly fluorescent compound.
Oxidation
By oxidation and hydroxylation epinephrine forms strongly fluorescing compound.
Epinephrine→ Highly fluorescent compound.
Thiamine is not itself fluorescent ,but it`s oxidation product thiochrome is fluorescent.
Thiamine → Thiochorme
Instrumentation
In fluorometry the intensity of radiation emitted as fluorescence related to the concentration of the
fluorescing species is measured. The instrumentation is for measuring the intensity of fluorescence as a
function of the wavelength of the radiation.
The chief components are:
a) Light source
b) Filter (Primary Filter) /monochromater
c) Sample holder
d) A emission filter (Secondary Filter) / emission
monochromater
e) Detector
f) Recorder and Amplifier
Md.
Imran
Nur
Manik
12. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 11
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Radiation source:
To produce exciting light, radiation source is required. The radiation source must be intense and stable.
Mercury arc and Xenon arc lamp are commonly used.
The emission of a mercury lamp is concentrated in several very intense bands. Among those having a wavelength
of 254-365 nm are of a great value as excitation radiation is evenly distributed over a wide range of wavelengths.
Md.
Imran
Nur
Manik
13. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 12
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Excitation filter:
It filters the source light and isolates the band of exciting light that is to be passed to the sample holder.
If the instrument uses coarse monochromator then the instrument is called fluorometer. If grating or
prism monochromator is used then the instrument is called spectrofluorometer,spectrophotofluorometer
or florescence spectrometers. Usually glass filters are used.
Sample holder:
Glass cells are used for most analysis. If measurement is to be under 320nm wavelength then quartz
cells are used.
Emission filter:
It selects the band of fluorescence which is to be detected. It is usually placed at right angle (90º) to the
beam of exciting (transmitting) light but other arrangements are possible.
Detector:
A photomultiplier and phototube is used to detect the fluorescent light and amplify it.
(The detector is placed at a right angle to the direction of travel of beam of exciting light.)
Recorder:
The output of the detector is connected to a meter, a digital display or a recorder. Recorder gives the
intensity of radiation in terms of electrical signal produced by the detector.
Factors influencing intensity of fluorescence
1. Concentration of fluorescing species
2. Presence of other solutes or impurities
3. pH of the sample solution
4. Stability of the sample compound
5. Solvent effects
6. Temperature
Mirror image rule
Vibrational levels in the excited states and ground states are similar.
An absorption spectrum reflects the vibrational levels of the electronically excited state.
An emission spectrum reflects the vibrational levels of the electronic ground state.
Fluorescence emission spectrum is mirror image of absorption spectrum.
Md.
Imran
Nur
Manik
14. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 13
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
1. Concentration of fluorescing species:
Fluorescence intensity (F) can be described as follows –
1).........(I)-(IkF
I)-(IF
So,
I)-(IF
and,
F
0
0
0
constantalityProportionk
lightedtransmitttheofIntensityI
lightincidenttheofIntensityI
efficiencyQuantum
intensityceFluorescenF
Here,
0
The exponential form of the Beer’s law (Beer-Lambert law) is
).(2..........eII εbc
0
solutionincompoundsampleofionConcentratc
lengthpathb
lightincidentofhwavelengtat thecompoundoftyabsorptiviMolar
Here,
By putting the value of I from equation (2) in equation (1) we get –
Thus we can see that the relationship between fluorescence intensity and concentration is quite
complex. But from the above equation,
When c increases,
εbc
e
1
value decreases and thus F value increases. So we can say that fluorescence
will increase with increase of concentration of fluorescing species.
lower conc.
Medium conc.
High conc.
Conc.
F
Sharp change
Sharpness decreases
Practically constant
Concentration reversal:
Concentration reversal is the phenomenon where the fluorescence intensity decreases as a result of
increase in concentration.
For some chemical species, if the supplied energy is fixed but the concentration is increased gradually
then at one point the fluorescence will decrease. This is because; the supplied energy fails to excite all
the molecules present in the solution at a time. So when the excited molecules emit energy (this is the
fluorescence), the previously unexcited molecules will absorb that energy. The emitted energy measured
is less i.e. the fluorescence intensity is less.
-εbc
0 0
-εbc
0
εbc
F = k (I - I e )
F = k I (1- e )
1
F (1 - )
e
Md.
Imran
Nur
Manik
15. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 14
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Conc.
F
Figure: Conc. reversal
For example the supplied energy can excite 20 molecules. If 25 molecules are present in the
solution then, the energy will excite 20 molecules and 5 molecules will remain unexcited. When the
excited molecules emit energy the unexcited molecules will absorb that energy.
However, if the sample is concentrated, sufficient light absorption might occur so that the portion sensed by
the detector is only weakly irradiated. This results in the phenomenon of concentration reversal.
2. Presence of other solutes/impurities:
A. Fluorescent impurities:
The sample solution may contain components other than the sample which is fluorescent. These
interfere with accurate measurement of fluorescence of the sample compound. Thus precautions such
as use of pure solvent and chemical reagents, cleanliness in all operations should be taken.
B. Inner-filter effect:
It is the reduction in the fluorescence intensity due to presence of non-fluorescent solutes which retard
penetration of light to or from fluorescent molecules.
Non-fluorescent molecules either prevent incident light from reaching the fluorescent molecules
(absorption retardation) or prevent emitted light detection.
Remedy: the non-fluorescent absorber must be eliminated or be maintained constant from sample to sample and a standard curve must
be used which was determined at that concentration of absorber. Or, the wavelength of excitation or emission radiation to minimize
this effect.
C. Chemical quenching:
Chemical quenching is the decrease of fluorescence intensity due to presence of any chemical in the
sample solution. i.e.
It is a chemical process where a chemical species reduce fluorescence intensity.
The chemical responsible for quenching is called quencher.
There are two types of quenching –
a. Collisional quenching: When the quencher absorbs the energy emitted by the excited fluorescent
molecules, it is called collisional quenching. Halide ions e.g. iodide, chloride ions cause it.
A molecule of “quencher" interacts with an excited molecule of the potentially fluorescing substance. Interaction results in the
dissipation of excitation energy not by fluorescence but by transfer of energy to the quenching molecule.
b. Static quenching: When the quencher absorbs the incident light in place of fluorescent
molecules, it is called static quenching. Xanthines (caffeine) and purines cause it for vitamin B12.
Md.
Imran
Nur
Manik
16. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 15
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
F
(ground state)
F*
(excited state)
F + Fluorescence
Quencher, Q
Q + F* Q* + F
Quencher, Q
Q* + F
Static quenching
Collisional quenching
Supplied energy
(incident light)
(Transmitted light)
+
3. pH of the sample solution:
Intensity of fluorescence is dependent upon the pH of the solution. This is due to two reasons –
A. Degree of ionization: In weak electrolytes pH affect the degree of ionization. Now, ionized and
unionized species may have different fluorescence intensity. Again, it is possible that ionized
species is fluorescent but the unionized species is not and vice versa. Thus pH may affect the
fluorescence intensity of a compound.
Exemplary, 2-naphthol ( 5.9apK ) shows fluorescence in both ionized and unionized forms. But
ionized form give fluorescence peak at 429µm whereas unionized form gives fluorescence peak at
359µm. So, if we measure fluorescence at 429µm (actually a filter is used to omit radiation below
415µm) then only the fluorescence of ionized species can be detected; this is detected at pH 8.5 and
above [Degree of ionization is detectable at pH equal to 1)(pKa ].
B. Excited-state dissociation: Sometimes, it is possible that a compound has different acid strength
in ground state and in excited state. So, if the excited state acid strength is greater, then the
compound will dissociate more easily when in excited state. Then, difference in the fluorescence
intensities of ionized and unionized species will cause change in fluorescence.
Exemplary, when fluorescence is measured at 429µm (a filter is used to omit radiation of below 415µm)
fluorescence is detected in the pH range of 2-8.5. But ground state 2-naphthol undergoes detectable
ionization at pH 8.5 and above. Excited state 2-naphthol underwent ionization in the pH range 2-8.5,
which is why we get fluorescence in that range.
4. Stability of the sample compound (Degradation of Sample)
If the compound being analysed is unstable in the experimental condition then fluorescence intensity will
change. Causes of instability may be due to –
Solvolytic degradation
Auto oxidation
Photo decomposition
Chemical degradation
Photodecomposition can be reduced by decreasing the intensity of the incident light. Also all
measurements should be completed as quickly as possible to avoid above problems.
Md.
Imran
Nur
Manik
17. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 16
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
5. Solvent effect:
Presence of impurities
Fluorescing impurities
Quenching impurities e.g. oxygen
Polarization effect
Quenching effect
Hydrogen bonding with analyte compound
6. Temperature:
Temperature reduces the fluorescence intensity. This is because of –
A. Increased internal conversion
Increased
temperature
Thermal motion of
molecules increased
Chances of intermolecular
collision is increased
Internal
conversion
Radiation
decreased
Fluorescence
decreases
B. Reduction in vibrational relaxation
In general, a 1⁰C rise in temperature results in a decrease of fluorescence intensity by 1%.
Comparison of Fluorometry with spectrophotometry
Sensitivity: Fluorometry is significantly more sensitive as an analytical tool than spectrophotometry.
The points included are.
In fuorometry the intensity of fluoresced light is measured directly by a fluorometer.
In spectrophotometry the intensity of light transmitted by a sample is measured and compared to that
transmitted by a blank.
The directly measured intensity can be amplified more readily and accurately in fuorometry than
the intensity difference measured in spectrophotometry.
In case of spectrophotometry the lower limit of detectability is determined by the smallest
concentration that will yield a detectable intensity difference between sample and blank. Here
small errors made in measuring the difference between the two intensities result in large errors
in calculated concentration.
The lowest limit of conc. that can be detected with accuracy is established by the molar
absorptivity in spectrophotometry (12mg/dl). The lower limit of conc. in fluorometry is
established by characteristics of the instrument and not usually by characteristics of the following
species.
Fluorescence measurements can offers sensitivity increases of 103-104 over absorbance
measurements.
Specificity:
Fluorometric assay can offer a degree of specificity that might not be attainable with a corresponding
spectrophotometric technique. The equations relating fluorescence intensity to concentration hold for
any region of the spectrum.
Md.
Imran
Nur
Manik
18. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 17
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Experimental Variables:
There are large no of experimental variable that must be controlled in fluorometric methods of analysis
than in corresponding spectrophotometric methods.
The temperature and the intensity of incident light must be maintained reasonably constant in a
fluorometric method, but not in a spectrophotometric procedure.
Extraneous solutes can markedly affect the intensity of fluorescence by quenching affects, but it
does not happen in spectrophotometry.
The influence of pH on fluorescence can be more complex than on absorbance and might
necessitate closer control of pH in fluorometric procedures than in spectrophotometric assays.
Difference between Absorption spectroscopy and Fluoroscence spectroscopy
Features Absorption spectroscopy Fluoroscence spectroscopy
Theoretical
consideration
Measurement of amount of light
absorbed.
Measurement of intensity of
fluorescence.
Wavelength of light
used
Which gives maximum absorption. Which gives maximum fluorescence.
Instruments
Determines only the absorption of
light.
Determines absorption of light as well
as emission of radiation.
Light source Tungsten, H2-discharge lamp. Mercury arc lamp, Xenon arc lamp.
Cell used Silica cell. Glass and metal cells.
Detector
Phototube or photo multiplier is used
to detect the radiation absorbed
Emission filter is used to separate the
emitted light from the transmitted light.
Concentration
Concentration depends on the molar
absorptivity.
Concentration depends on the
characteristics of the instrument.
Electrical transition
Applicable for both ππ* & nπ*
transition.
Not applicable for the compound
containing nπ* transition.
Experimental
variables
temperature &
Extraneous solution
Not so restricted. Highly restricted.
Sensitivity and
selectivity
Less sensitive and less specific. More sensitive and highly specific.
Applications of fluorometry
Application in Chemistry:
Fluorometry is used in chemistry for –
1. Determination of metal ions: Complexes of metals ions may give strong fluorescence which is
utilized for this purpose.
2. Separation and identification: In many cases, after separation, chemicals are identified using
fluorometry. e.g. aminocrine.
Md.
Imran
Nur
Manik
19. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 18
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Application in Biopharmaceutics:
1. Measurement of drug in blood, urine and other body fluids.
2. Study of the rate and mechanism of drug absorption, metabolism and excretion.
3. Selection of toxic compounds.
Pharmaceutical applications:
Fluorometry is used for quantitative analysis of –
1. Hormones: Adrenaline, aldosterone, testosterone
2. Alkaloids:
a. Opioids: Morphine, codeine etc.
b. Rauwolfia alkaloids: Reserpine
c. Others: Atropine, emetine etc.
3. Vitamin: Riboflavin and thiamine are indicated for fluorometric assay by USP and BP.
4. Antibiotics: tetracycline, sulfonamide etc.
5. Cardiac glycosides: Such as digoxin, digitoxin, etc.
Fluorometry is also used for qualitative analysis of these drugs.
6. Fluerometry is widely used in the analysis of drugs in systems (physiological systems) other than
dosage forms. The sensitivity of the method of analysis is applied for a large number of
pharmacological, biochemical, toxicological, pharmacokinetic (ADME) & biopharmaceutical
studies for the analysis of amount of drugs in biological fluids and tissues.
Advantages of fluorometry
1. Sensitivity: In case of Fluorescence, detectability to parts per billion or even parts per trillion is
common for most analytes. This extraordinary sensitivity allows the reliable detection of fluorescent
materials (chlorophyll, aromatic hydrocarbons, etc.) using small sample sizes. Also, field studies can be
performed in open waters without sample treatment. Fluorometers achieve 1,000 to 500,000 times
better limits of detection as compared to spectrophotometers.
2. Specificity: Spectrophotometers merely measure absorbed light and as many materials absorb light, it
becomes difficult to isolate the targeted analyte in a complex matrix. Fluorometers are highly specific
and less susceptible to interferences because fewer materials absorb and also emit light (fluoresce).
And, if non-target compounds do absorb and emit light, it is rare that they will emit the same wavelength of light as
target compounds.
3. Wide Concentration Range: Fluorescence output is linear to sample concentration over a very broad
range. Fluorometry can be used over three to six decades of concentration without sample dilution or
modification of the sample cell.
6. Simplicity and Speed: Fluorometry is a relatively simple analytical technique. Fluorometry's sensitivity
and specificity reduce or eliminate the sample preparation procedures often required to concentrate
analytes or remove interferences from samples prior to analysis. This reduction in or elimination of
sample preparation time not only simplifies, but also expedites the analysis.
7 Low Cost: Reagent and instrumentation costs are low when compared to many other analytical
techniques, such as gas chromatography and HPLC.
Reagent costs are low because, due to the high sensitivity of fluorometers, fewer reagents can be used. And,
small laboratory filter fluorometers can now be purchased for less than $3,000 USD.
Md.
Imran
Nur
Manik
20. Fluorometry
Prepared By: Md. Imran Nur Manik; B.Pharm; M.Pharm Page 19
manikrupharmacy@gmail.com; Lecturer; Department of Pharmacy; Northern University Bangladesh.
Limitations of Fluorometry
1. Molecules should be fluorescent to measure by the fluorescence spectroscopy.
2. All kind of materials on substances cannot be detected by it.
References
Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York.
Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York. 3 Id., p. 7.
Dr. Richard Thompson. 1998. University of Maryland, Department of Biochemistry and Molecular
Biology, School of Medicine.
G. K. Turner, "Measurement of Light From Chemical or Biochemical Reactions," in Bioluminescence
and Chemiluminescence: Instruments and Applications, Vol. I, K. Van Dyke, Ed. (CRC Press, Boca
Raton, FL, 1985), pp. 45-47.
Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, pp. 51-57.
Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York, chap. 2.
Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, pp. 67-69.
Lakowicz, J.R. 1983. Principles of Fluorescence Spectroscopy, Plenum Press, New York, pp. 23-26.
Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, pp. 57-58.
Stotlar, S. C. 1997. The Photonics Design and Applications Handbook, 43rd Edition, Laurin Publishing
Co., Inc., Pittsfield, MA, p. 119.
Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, p. 63.
Dr. Richard Thompson. 1998. University of Maryland, Department of Biochemistry and Molecular
Biology, School of Medicine.
Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, p. 30.
Dr. Richard Thompson. 1998. University of Maryland, Department of Biochemistry and Molecular
Biology, School of Medicine.
Iain Johnson, Product Manager, and Ian Clements, Technical Assistant Specialist (May 1998
communication from Molecular Probes, Eugene, Oregon).
Fluorometric Facts: A Practical Guide to Flow Measurement, Turner Designs (1990), pp. 14-15.
Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York, p. 172.
Fluorometric Facts: A Practical Guide to Flow Measurement, Turner Designs (1990), p. 21.
Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York., p. 28.
Teitz Textbook of Clinical Chemistry and Molecular diagnosis (5th Edition)
Dr.B.K.Sharma, Instrumental methods of chemical analysis.
Gurdeep R Chatwal, Instrumental methods of chemical analysis
http://en.wikipedia.org/wiki/Fluorescence
http://images.google.co.in/imghp?oe=UTF-8&hl=en&tab=wi&q=fluorescence
http://www.bertholdtech.com/ww/en pub/bioanalytik/biomethods/fluor.cfm
Md.
Imran
Nur
Manik