Name: fluorescent and phosphorescent analysis
Uploaded: 2024-12-05T18:32:05.000Z
Duration: 1 min 14 s
Channel: JoVE

Fluorescence and phosphorescence are among the most sensitive analytical techniques due to their lower detection limits than absorption spectrophotometry.
Non-photoluminescent inorganic analytes are determined indirectly by forming a fluorescent metal-ligand complex with an organic ligand or by measuring a decrease in fluorescence upon adding the analyte to a solution containing a fluorescent molecule.
Organic compounds with aromatic rings are typically fluorescent, while aromatic heterocycles often display phosphorescence.
Non-photoluminescent organic analytes may be incorporated into reactions producing fluorescent or phosphorescent products.
Phosphorescence and fluorescence methods are complementary, as strongly fluorescing compounds often exhibit weak phosphorescence and vice versa.
Phosphorimetry is not as widely used as fluorimetry due to low-temperature requirements and low precision.
Recently developed room-temperature phosphorimetric methods involve binding the analyte to a solid support like filter paper or incorporating the analyte into micelle cores for protecting the triplet state.&#160;

photoluminescence: applications

Photoluminescence: Applications

Photoluminescence offers a wide range of applications due to its inherent sensitivity and selectivity. This technique allows for both direct and indirect analyses of the analyte. Direct quantitative analysis is possible when the analyte exhibits a favorable quantum yield for fluorescence or phosphorescence. However, an indirect analysis may be feasible if the analyte is not fluorescent or phosphorescent, or if the quantum yield is unfavorable. Indirect methods include reacting the analyte with a reagent to form a fluorescent product or measuring the decrease in fluorescence upon adding the analyte to a solution containing a fluorescent molecule.
Inorganic ions, with a few exceptions like UO2+, are generally not sufficiently fluorescent for direct analysis. These ions can be analyzed with an organic ligand to form a fluorescent or phosphorescent metal-ligand complex. Organic compounds containing aromatic rings are typically fluorescent, while aromatic heterocycles tend to be phosphorescent. If the organic analyte is not naturally fluorescent or phosphorescent, it can sometimes be incorporated into a chemical reaction to produce a fluorescent or phosphorescent product. For instance, the enzyme creatine phosphokinase can be determined by catalyzing the formation of creatine from phosphocreatine, which then reacts with ninhydrin to yield a fluorescent product of unknown structure.
Phosphorescence and fluorescence methods are complementary as strongly fluorescing compounds exhibit weak phosphorescence and vice versa. Phosphorimetry has been used to determine a variety of organic and biochemical species. However, it is not as widespread as fluorometry, possibly due to the need for low temperatures and the generally poorer precision of phosphorescence measurements. In recent years, considerable effort has been put into developing phosphorimetric methods that can be carried out at room temperature. These include methods where the analyte is bound to a solid support, such as filter paper or silica gel, or incorporated into the core of micelles or cyclodextrin molecules. In most room-temperature experiments, heavy atoms, such as Tl(I), Pb(II), Ag(I), and halide ions, are used to promote intersystem crossing.

Photoluminescence offers a wide range of applications due to its inherent sensitivity and selectivity. This technique allows for both direct and indirect ...

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Analytical Chemistry

Unit 1

Introduction to Molecular Spectroscopy

KEY TERMS AND CONCEPTS

fluorescent and phosphorescent analysis

Photoluminescence is useful in chemical analysis because it is sensitive and selective. It can support both direct and indirect measurements of an analyte, which is the substance being tested. When an analyte has a strong quantum yield, it can be measured directly by fluorescence or phosphorescence.

Indirect analysis is used when the analyte is not naturally fluorescent or phosphorescent, or when its quantum yield is poor. One approach is to react the analyte with a reagent to make a fluorescent product. Another is to measure how much fluorescence drops after the analyte is added to a solution that already contains a fluorescent molecule.

Inorganic ions usually do not fluoresce enough for direct analysis, with a few exceptions such as UO2+. These ions can often be measured by forming a fluorescent or phosphorescent metal-ligand complex with an organic ligand. Many organic compounds with aromatic rings are fluorescent, while aromatic heterocycles tend to be phosphorescent.

If an organic analyte is not naturally fluorescent or phosphorescent, it may be built into a reaction that creates a luminescent product. A common example is creatine phosphokinase, which is measured by forming creatine from phosphocreatine. The creatine then reacts with ninhydrin to produce a fluorescent product of unknown structure.

Fluorescence and phosphorescence are complementary methods. Strongly fluorescent compounds usually show weak phosphorescence, and strongly phosphorescent compounds usually fluoresce weakly. Phosphorimetry has been used for many organic and biochemical species, but it is less common than fluorometry because it often needs low temperatures and gives poorer precision.

Recent work has focused on phosphorimetric methods that work at room temperature. Some use a solid support such as filter paper or silica gel. Others place the analyte inside micelles or cyclodextrin molecules. In many of these methods, heavy atoms such as Tl(I), Pb(II), Ag(I), and halide ions help promote intersystem crossing.

Fluorescent and Phosphorescent Analysis

dual nature of electromagnetic (em) radiation

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EM Spectrum, Waves, and Photon Energy

Electromagnetic radiation has both wave and particle features. It includes electric and magnetic field components that oscillate in planes perpendicular to each other. These fields are also perpendicular to the direction the radiation moves through space. As a wave, electromagnetic radiation is described by wavelength and frequency. Wavelength is the distance between two consecutive peaks or troughs. Frequency is the number of wave cycles that pass a point in a fixed time. The electromagnetic...

interaction of em radiation with matter: spectroscopy

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EM Radiation and Matter in Spectroscopy

Electromagnetic radiation and matter interact in ways that reveal details about atoms and molecules. In spectroscopy, scientists study how EM radiation is absorbed, emitted, transmitted, reflected, or scattered by a substance. This helps identify its chemical composition and properties. EM radiation behaves as both a wave and a particle. As a wave, it has intensity and amplitude. Intensity, also called irradiance, is the number of photons that strike a given area in a certain time. Amplitude...

molecular spectroscopy: absorption and emission

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Molecular Energy Levels in Spectroscopy

Molecular spectroscopy describes how molecules absorb and emit light by moving between energy levels. These levels are called quantum states. Unlike atoms, molecules also have rotational and vibrational energy levels, so their energy structure is more complex. Each level is separated by an energy gap. The gaps between electronic, vibrational, and rotational levels are very different in size. A diatomic molecule can be used to show these three kinds of energy levels. A molecule can absorb a...

spectrophotometry: introduction

Spectrophotometry for Absorption Spectra

Spectrophotometry measures how a material absorbs, reflects, diffracts, or transmits electromagnetic radiation. It compares the intensity of radiation before and after it interacts with the material. The result shows how the material responds to different wavelengths. A spectrophotometer is the device used for this measurement. It includes a source of electromagnetic radiation, a slot for the sample, and a detector. The detector measures the radiation that leaves the material, including...

ultraviolet and visible (uv&ndash;vis) spectroscopy: overview

ultraviolet-and-visible-uvvis-spectroscopy-overview

UV-Vis Spectroscopy for Light Absorption

UV-Vis spectroscopy measures how materials absorb ultraviolet and visible light. It is a useful analytical method because it is versatile, simple to use, and gives results quickly. Scientists use it for both qualitative and quantitative analysis. When UV-Vis radiation passes through a sample, molecules absorb specific wavelengths based on the energy needed for electronic transitions. This absorption moves atoms or molecules from a ground state to an excited state. If a molecule absorbs UV...

uv&ndash;vis spectroscopy: molecular electronic transitions

uvvis-spectroscopy-molecular-electronic-transitions

UV-Vis Spectroscopy and Molecular Absorption

UV-Vis spectroscopy shows how molecules absorb ultraviolet and visible light. It is used to study electronic structure by tracking how electrons move between molecular orbitals. The absorbed light must match the energy gap between the ground state and an excited state. In this process, an electron moves from the highest occupied molecular orbital, or HOMO, to the lowest unoccupied molecular orbital, or LUMO. This jump raises the electron to a higher energy level. The energy difference between...

uv&ndash;vis spectrometers

UV-Vis Spectrophotometer Parts and Samples

A UV-visible spectrophotometer measures how much UV and visible light a sample absorbs. It uses a light source, a wavelength selector, a sample holder, and a detector to build the spectrum. This makes it useful for checking liquid samples in the lab. The light source depends on the region being measured. A deuterium lamp provides UV radiation, and a tungsten lamp provides visible radiation. A monochromator or prism acts as a diffraction grating and splits the incoming light into different...

uv&ndash;vis spectrum

How Molecular Structure Shapes UV-Vis Bands

UV-Vis spectroscopy shows how a molecule absorbs light in the ultraviolet and visible ranges. When light passes through a substance, some of it is absorbed and the rest is reflected or transmitted. Molecules that absorb between 180 and 400 nm give a UV spectrum, while absorption in the 400 to 780 nm range gives a visible spectrum. A UV-Vis spectrum is a plot of absorbance versus wavelength. The y-axis shows molar absorptivity, ε, or log ε, and the x-axis shows wavelength. Absorbance describes...

uv&ndash;vis spectroscopy: beer&ndash;lambert law

Using Absorbance to Find Concentration

The Beer-Lambert law links absorbance, concentration, and path length in a solution. It is based on ideas from Johann Heinrich Lambert and August Beer. Their work helps explain how light changes as it passes through a medium. Lambert's law says that when light passes through a medium, the loss in intensity is directly proportional to the original intensity and the path length. Beer’s law adds that the transmittance of a solution stays constant if the product of concentration and path length...

photoluminescence: fluorescence and phosphorescence

photoluminescence-fluorescence-and-phosphorescence

Light Emission from Excited Molecules

Photoluminescence is light emission from molecules after they absorb light energy. A substance takes in photons, which raises electrons to excited energy levels. The electrons then relax back to the ground state and release light. This process appears in many materials, including semiconductors, organic compounds, and inorganic compounds. The main role of electron spin helps separate photoluminescence into fluorescence and phosphorescence. In a singlet spin state, two electrons share the same...

variables affecting phosphorescence and fluorescence

variables-affecting-phosphorescence-and-fluorescence

Factors That Change Light Emission in Molecules

Fluorescence and phosphorescence depend on several factors that change how molecules emit light. These effects matter in analytical chemistry, biological imaging, and materials science because they help scientists detect molecular properties and see cellular structures. Molecular structure is one of the main influences. Aromatic compounds with low-energy pi-bonding to pi-non-bonding transitions often give strong fluorescence. Aliphatic and alicyclic carbonyl compounds, as well as highly...

deactivation processes: jablonski diagram

Jablonski Diagram: Light Loss Pathways

Jablonski diagrams show how excited molecules lose energy after absorbing light. They are used to track both light-emitting and non-light-emitting steps in luminescence. The diagram gives a simple picture of the energy states a molecule can move through. The lowest line in a Jablonski diagram is the ground-state energy of the molecule. This state is usually a singlet state, and it represents the condition of most molecules in solution at room temperature. The lines above it show vibrational...

Photoluminescence is useful in chemical analysis because it is sensitive and selective. It can support both direct and indirect measurements of an analyte, which is the substance being tested. When an analyte has a strong quantum yield, it can be measured directly by fluorescence or phosphorescence. Indirect analysis is used when the analyte is not naturally fluorescent or phosphorescent, or when its quantum yield is poor. One approach is to react the analyte with a reagent to make a...

fluorescence and phosphorescence: instrumentation

fluorescence-and-phosphorescence-instrumentation

Measuring Fluorescence and Phosphorescence

Fluorometers and spectrofluorometers are instruments used to measure molecular fluorescence. They do this by choosing specific excitation and emission wavelengths. The two instruments use different light sources and wavelength-selection methods. A fluorometer uses absorption interference filters to select the excitation and emission wavelengths. Its excitation source is usually a low-pressure mercury vapor lamp. This lamp gives intense lines across the ultraviolet and visible regions.

key terms and concepts

Explore how fluorescence and phosphorescence are used to analyze inorganic ions, organic compounds, and biochemical species with direct and indirect methods.

Fluorescent and Phosphorescent Analysis | JoVE K12

analytical chemistry

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Fluorescent and Phosphorescent Analysis

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