FLUORIMETRY by g.v.durgamani
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FLUORIMETRY introduction and easy concept of Fluorimerty instrumentation and application of fluorimetry
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Flourimetry
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.
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The document describes the principles and components of flame photometry. Flame photometry measures the intensity of light emitted from metal atoms excited by the heat of a flame. When a solution is sprayed into the flame, the solvent evaporates and the metal atoms are excited and emit light of characteristic wavelengths. A mirror collects the light, which is separated into its wavelengths by a prism or grating. A photodetector measures the light intensities, which correspond to concentrations of metals in the original solution. Common applications include analyzing body fluids, soils, and water.
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There are two major factors that affect fluorescence intensity: the intrinsic structure of a molecule and the environment of a molecule. The intrinsic structure, such as conjugation, aromaticity, and substituent groups, determines a molecule's ability to fluoresce. The environment, like temperature, viscosity, oxygen levels, solvent polarity, pH, light exposure, and concentration, can impact fluorescence through molecular collisions and interactions. Understanding how both the intrinsic and external factors influence fluorescence is important for quantitative fluorescence applications.
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Spectrofluorimetry is a technique that measures fluorescence emitted from molecules. It involves exciting molecules with UV or visible light which causes electrons to transition to an excited state. The molecule then relaxes and emits light of a longer wavelength. Factors like concentration, quantum yield, path length, pH, temperature and presence of quenchers affect the intensity of fluorescence. Spectrofluorimeters are used to collect excitation and emission spectra of molecules to identify them.
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The document discusses spectrofluorimetry and luminescence spectroscopy. It defines fluorescence and phosphorescence as types of photoluminescence that occur when a molecule absorbs radiation and then emits light as it relaxes to the ground state. Fluorescence emission occurs from the lowest excited singlet state on a timescale of 10-9 to 10-7 seconds, while phosphorescence emission occurs from the lowest triplet excited state on a longer timescale of 10-6 to 10 seconds. The document also provides examples of applications including the analysis of polyaromatic hydrocarbons like benzo(a)pyrene and fluorimetric drug analysis including the detection of LSD.
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Nmr spectroscopy
1. 1H NMR spectroscopy is a technique used to analyze compounds by detecting hydrogen nuclei in a magnetic field. It provides information about functional groups, number of nuclei, and structure of compounds.
2. The principle involves hydrogen nuclei absorbing radio frequencies matching their Larmor frequency in an applied magnetic field. This absorption is measured to produce an NMR spectrum.
3. Factors like electronegativity, magnetic anisotropy, and spin-spin coupling influence the chemical shifts observed on the NMR spectrum, allowing identification of functional groups and structure elucidation.
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In this slide contains principle and types of detectors used in HPLC.
Presented by: K.Tarun. (Department of industrial pharmacy).
RIPER, anantpur.
IR Spectroscopy - Sudheerkumar Kamarapu, M. Pharmacy Lecture pdf
Infrared spectroscopy (IR) measures the bond vibration
frequencies in a molecule and is used to determine the
functional groups.
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Mass spectrometry deals with studying charged molecules and fragment ions produced from a sample exposed to ionizing conditions. It provides the relative intensity spectrum based on ions' mass to charge ratio, allowing identification of unknown compounds. The document discusses the basic principles, advantages, disadvantages, instrumentation, applications, and analysis techniques of mass spectrometry.
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Fluorimetry is a technique that measures fluorescence intensity at a particular wavelength using a fluorimeter or spectrofluorimeter. It works by exciting a molecule's electrons with radiation, causing them to emit radiation upon returning to the ground state. Factors like concentration, quantum yield, incident light intensity, pH, temperature and presence of quenchers can affect fluorescence. Fluorimetry has advantages like high sensitivity, precision and specificity and is useful for determining various inorganic/organic substances and compounds.
Infrared spectroscopy
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IR Spectroscopy - Sudheerkumar Kamarapu, M. Pharmacy Lecture pdf
Ir spectroscopy instrumentation, b y -dr. umesh kumar sharma and arathy s a
Ir spectroscopy instrumentation, b y -dr. umesh kumar sharma and arathy s a
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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.
Fluorimetry
Fluorimetry involves measuring the fluorescence light emitted by a substance. A fluorimeter is used, which contains a light source, filters or monochromators to select excitation and emission wavelengths, sample cells, and a detector. Fluorescence occurs when molecules absorb light and emit light of a longer wavelength as they transition from an excited singlet state to the ground state. Factors like concentration, pH, and temperature can affect fluorescence. Fluorimetry is used in applications like determining ions and vitamins, and in organic analysis.
Flourimetry 140618015916-phpapp01 - copy
This document discusses fluorescence spectroscopy and its applications in pharmacy. It begins with definitions of fluorescence, phosphorescence, and chemiluminescence. It describes how fluorescent substances emit light when exposed to radiation and discusses factors that affect fluorescence like molecular structure, substituents, concentration, oxygen, pH, and temperature. The principles of fluorescence are explained using Jablonski diagrams. Instrumentation for fluorescence spectroscopy including light sources, filters, sample cells, and detectors are outlined. Finally, applications of fluorescence spectroscopy in inorganic analysis, organic analysis, liquid chromatography, and determination of vitamins and drugs are described.
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FLUORIMETRY by g.v.durgamani
- 1. FLUORIMETRY Prepared by G.V.Durgamani B . Pharmacy 7th sem. 1803268032
- 2. FLUORIMETRY ; An analytical method for detecting and measuring fluorescence in compound or target such as cell, proteins or nucleotides or targets previously labeled which fluorescence agents. Luminescence; it is the emission of light by a substance .It occurs when an electron returns to the electronic ground state from an excited state and loses its excess energy as a photon. It is of two types; Fluorescence spectroscopy Phosphorescence spectroscopy INSTRUCTIONS
- 3. 1 When a beam of light is incident on certain substances they emit visible light or radiations this is known as fluorescence 2 Fluorescence starts immediately after the absorption of light and stops as soon as the incident light is cut off. 3 The substances this phenomen are known as flourescent substances 4 Time required for compound to show fluorescence is 10-6 to 10-4 . FLUORESCENCE
- 4. When light rdiation is incident on certhain substances they emmit light continuously even after the incident light is cut off. 01 This type of delayed fluorescence is called phosphorescence. 02 Substance showing phosphorescence are phosphorescencent substance. 03 Time required for compound to show phosphorescence is 10-4 to 20s . 04 PHOSPHORESCENCE
- 5. The wave length of emmited radiation is longer then the absorbed radiation. i.Stoke’s The wave length of emmited radiation shorter then the absorbed radiation. ii.Empty stoke’s When the wave length of immited radiationis equal to the absorbed radiation. iii.Resonance When the elements like Thallium ,zinc ,Cadmium or an Alkali metal are added to the Mercury vapor these elements are sensitized and give fluorescence. i.Sentized The excitation is partially by EMR and partially by thermal energy. ii.Thermal assisted Fluorescence Based on wave length Based on phenomenon
- 6. o A molecular electronic state in which all of the electrons are paired are called singlet state. o In a singlet state molecules are diamagnetic. o Most of the molecules in their ground state are paired. ● When such a molecule absorbs UV/Visible radiation, one or more of the paired electron raised to an excited singlet/excited triplet state. ● The process of promotion of electron from HOMO to LUMO with absorption of energy is called as excitation. PRINCIPLE
- 7. Nature of molecule Concentration Quantum yield Intensity of light pH Oxygen Temperature Viscosity Factors affecting Fluorescence
- 9. Applications fluorescence 1.Determination of uranium salts and this is used extensively in the field of nuclear research. 2. determination of inorganic ions. 3.fluorescencent indicator. 4.determination of organic substances. 5.pharmaceutical applications.
- 10. Thank you Reference; Textbook of Instrumental Method of Analysis by Dr.Abhishek Tiwari