Fluorescence Spectrophotometry - Definition, Principle, Parts, Advantages, Uses

Q: What is Fluorescence Spectrophotometry?

Fluorescence Spectrophotometry is a technique used to measure the intensity of light emitted by a substance when it is excited by a specific wavelength of light.

Q: What is the principle of Fluorescence Spectrophotometry?

The principle of Fluorescence Spectrophotometry is based on the ability of certain molecules to absorb light at one wavelength and re-emit it at a longer wavelength.

Q: What are the key parts of a Fluorescence Spectrophotometer?

The key parts of a Fluorescence Spectrophotometer include a light source, a monochromator, a sample holder, a detector, and a readout device.

Q: What are the advantages of Fluorescence Spectrophotometry?

Some of the advantages of Fluorescence Spectrophotometry include high sensitivity, high selectivity, and the ability to measure very small amounts of a substance.

Q: What are some common uses of Fluorescence Spectrophotometry?

Common uses of Fluorescence Spectrophotometry include studying the structure of proteins and nucleic acids, monitoring chemical reactions, and measuring the concentration of analytes in a sample.

Q: What is the difference between Fluorescence Spectrophotometry and Absorption Spectrophotometry?

Fluorescence Spectrophotometry measures the light emitted by a substance after it has been excited by a specific wavelength of light, while Absorption Spectrophotometry measures the amount of light absorbed by a substance.

Q: Is Fluorescence Spectrophotometry harmful to the environment?

Fluorescence Spectrophotometry is not harmful to the environment as it uses small amounts of energy and does not produce any harmful by-products.

Q: How is Fluorescence Spectrophotometry used in medical research?

Fluorescence Spectrophotometry is used in medical research to study the structure and function of proteins and nucleic acids, and to monitor the effectiveness of drugs and other treatments.

Q: What are the most common Fluorescence Spectrophotometry instruments?

The most common Fluorescence Spectrophotometry instruments include Fluorometers, Fluorimeters, and Fluorescence Microscopes.

Q: Can Fluorescence Spectrophotometry be used to measure the concentration of analytes in real-time?

Yes, Fluorescence Spectrophotometry can be used to measure the concentration of analytes in real-time, making it a useful tool for monitoring chemical reactions and other processes.

The technique of fluorescence spectrophotometry measures the intensity of light emitted by a substance after it has been excited by a certain wavelength of light. This method is employed to investigate the characteristics of molecules and detect the presence of specific chemicals in a sample.

The origins of fluorescence spectrophotometry can be traced back to the early 20th century, when scientists began to investigate the phenomena of fluorescence. In the 1920s and 1930s, scientists such as George Van Nevel and Robert Boyle began to develop methods for measuring the fluorescence intensity, providing the groundwork for the present approach of fluorescence spectrophotometry.

Fluorescence spectrophotometry is an essential instrument in numerous disciplines, such as chemistry, biology, and medicine. It is used to research the characteristics of molecules and identify unknown substances in chemistry. It is used to examine the structure and function of proteins and other biomolecules in biology. In the medical field, it is used to detect the presence of disease indicators and investigate the interactions between medications and biological substances.

What is fluorescence spectrometry?

On the basis of an analyte’s fluorescent characteristics, fluorescence spectrometry is a quick, straightforward, and inexpensive approach for determining the concentration of the analyte in solution. When the type of compound to be examined (‘analyte’) is known, it can be utilised to do a quantitative analysis to determine the concentration of the analytes in very easy procedures. Fluorescence is mostly used to measure substances in solution.

In fluorescence spectroscopy, a beam with a wavelength ranging from 180 to 800 nm travels through a cuvette containing a solution. Then, we measure, at an angle, the light emitted by the sample. In fluorescence spectrometry, it is possible to measure both the excitation spectrum (the light absorbed by the sample) and the emission spectrum (the light released by the sample). The relationship between the concentration of the analyte and the intensity of the emission is direct.

There are various parameters that affect the spectrum’s intensity and shape. When capturing a spectrum of emission, the intensity is based on:

Optimal excitation wavelength
Concentration of the solvent of the analyte
Length of the cuvette’s path

Self-centeredness of the sample

Alzheimer’s Fluorescence and phosphorescence are processes of photon emission that occur during molecule relaxation from excited electronic states. These photonic processes include the transfer of polyatomic fluorescent compounds between electronic and vibrational states (fluorophores). Fluorophores are indispensable to fluorescence spectroscopy. Fluorophores are the molecular components responsible for fluorescence. The majority of fluorophores consist of molecules with aromatic rings, such as Tyrosine, Tryptophan, Fluorescein, etc. Luminescence, the non-thermal emission of light by a substance, is a form of cold body radiation. It may result from chemical reactions, electrical energy, subatomic vibrations, or crystal stress. Two prerequisites are necessary for luminescence:

The luminous material must be a semiconductor with a band gap greater than zero. [Metals are incapable of emitting light if they lack a band gap.]

Before luminescence can occur, this substance must be supplied with energy.

Fluorescence Spectrum | Image Source: www.horiba.com

Principle of fluorescence spectroscopy

Fluorescence is a sort of radiative emission that occurs when a molecule absorbs energy at a wavelength where it possesses a dipole moment of transition. The excitation energy supplied to the molecule in its ground state propels photons to an excited singlet state, from which they decay to the lowest vibrational energy level of this excited singlet state. As seen in Figure , this energy relaxes back to the ground state of the molecule, producing photons in the process.

Jablonski diagram illustrating different transitions between a molecule’s energy states.

There are three techniques of nonradiative relaxation for fluorescent molecules in which the excitation energy is not transformed into photons: (1) internal conversion, (2) external conversion, and (3) intersystem crossover. When there is a relatively small energy gap between two electronic states and electrons shift from a higher energy level to a lower energy one, internal conversion happens. Here, the energy is transmitted to the electronic state’s vibrational modes. Since vibrational processes are thermally driven, an increase in temperature diminishes the intensity of fluorescence. Energy is lost during external conversion due to collisional quenching with solute molecules in the fluorophore’s surroundings. Intersystem crossover occurs when the energy levels of the excited singlet and triplet states overlap and electrons transition from the lowest excited singlet state to the first excited triplet state. The emission of photons as electrons return to their ground state is known as phosphorescence. Because the triplet state has less energy than the singlet state, phosphorescence peaks occur at longer wavelengths than fluorescence peaks. Since these transitions are likewise prohibited, phosphorescence has a longer duration than fluorescence ((~10^-4 – 10^-2 seconds vs. ~10^-9 – 10^-6 seconds). Longer lives also result in thermal deactivation by means of oxygen quenching, solvent migration, and intermolecular collisions. Consequently, phosphorescence cannot normally be noticed at ambient temperature; samples must be cooled to liquid nitrogen temperature.

Beer’s Law and Concentration Effects

While absorption happens in less than 10 to 15 seconds, the transition from an aroused to a resting state is considerably slower. Therefore, unlike absorption, fluorescence can reveal information regarding a fluorophore’s interactions with surrounding molecules and solvents.

Fluorescence intensity is proportional to the intensity of the excitation light.

F=2.303 * K * I₀* εbc

where K is a constant determined by the geometry of the instrument, I0 is the intensity of the excitation light, e is the molar absorptivity of the fluorophore, b is the pathlength, and c is the concentration. Since the fluorescence intensity is not proportional to the incident light intensity, as is the case with absorption measurements, the fluorescence sensitivity is significantly higher because it is not constrained by the instrument’s ability to distinguish between the incident and detected intensities. Consequently, measurements require lesser concentrations.

The preceding equation is linear only when the absorbance of the sample is less than 0.05 AU. If a sample is overly concentrated, the emitted light may be reabsorbed by the fluorophore, resulting in a decrease in the fluorescence signal at shorter wavelengths. In addition, excitation light may not penetrate the whole width of a highly concentrated sample, resulting in diminished fluorescence intensities.

Fluorescence Phenomenon

There is a discrete succession of energy levels within every molecule. Absorption of a photon by a molecule causes an electron to be boosted from the ground state (S₀) to one of several excited singlet states (S₁, S₂, etc.).

Complex molecular systems comprising atoms with lone pairs of electrons, such as O and N, and aromatic and/or aliphatic conjugated unsaturated systems capable of a high degree of electron delocalization are particularly likely to undergo transitions of this type.
The electron is then dropped to the lowest vibrational level of the lowest excited state, S1, and energy is liberated by “internal conversion.”
Any excited state then relaxes down to its lowest vibrational level. Different methods can then be used to relax from the S1 state, which has a lifespan of the order of 10^-9 s, to the groundstate S0 .

These include: (1) Fluorescence, a radiative photoprocess in which energy is lost during internal conversion processes and results in the emission of photons that are less energetic than those absorbed; and (2) various nonradiative processes, including: (a) internal conversion processes occurring by relaxation through internal vibrations or through collisions with solvent and/or solute molecules; (b) transfer of energy to another chemical compound resulting in a photochemical reaction or formation of an excited-state dimer (excimer) or excited-state complex (exciplex) which emits photons at longer wavelengths than fluorescence; (c) intersystem crossover to a triplet state, T1, with a substantially longer lifetime (typically >10^-5s) than its S₁ precursor, which is followed by either a delayed release of energy known as the phosphorescence phenomena or by relaxation via internal conversion mechanisms.

The Electronic Excited State

Fluorescence and phosphorescence are processes of photon emission that occur during molecule relaxation from excited electronic states. These photonic processes include the transfer of polyatomic fluorescent compounds between electronic and vibrational states (fluorophores). The excited state structure and associated transitions are conveniently shown using the Jablonski diagram.
Typically, the energy separating electronic states are on the order of 10 000^-1 cm. Each electronic state is subdivided into a number of sublevels that correspond to the vibrational modes of the molecule.

The distance between the energies of the vibrational levels is approximately 100^-1cm. To cause an electronic transition, photons with energy in the ultraviolet to blue-green part of the spectrum are required.
In addition, because the energy gap between the excited and ground electronic states is substantially greater than the thermal energy, thermodynamics predicts that molecules primarily exist in the electronic ground state. Based on their multiplicity, the electronic excited states of a polyatomic molecule can be further classified.
The indistinguishability of electrons and the Pauli exclusion principle need symmetric or asymmetric spin states for the electronic wave functions. The symmetric wave functions, commonly known as the triple state, have a multiplicity of three, or three forms.

The antisymmetric wave function, often known as the singlet state, has a singular shape, one multiplicity. The optical transition couples states with the same multiplicity to the first order. The optical transition stimulates molecules from the lowest vibrational level of the electronic ground state to a vibrational level that is accessible in the electronic excited state.
Since the electronic state of the ground is a singlet, the electronic state of the destination is also a singlet. The molecule is rapidly relaxed to the lowest vibrational level of the excited electronic state following excitation. On the time scale of femtoseconds to picoseconds, this quick vibrational relaxation process happens.
The Stoke shift is the result of this relaxation procedure. The Stoke shift describes the observation that the wavelength of fluorescence photons is greater than that of excitation light. Fluorescence lifetime is a period on the order of nanoseconds during which the fluorophore maintains the lowest vibrational level of the excited electronic state.

Fluorescence emission happens when the singlet electronic excited states of the fluorophore decay to an acceptable vibrational level in the electronic ground state. The absorption and emission spectra of fluorescence represent the vibrational level structures of the ground and excited electronic states, respectively.
The Frank–Condon principle argues that electronic transitions do not significantly modify the vibrational levels. Because of the similarities between the vibrational level structures of the ground and excited electronic states, the absorption and emission spectra frequently exhibit mirrored characteristics.
The excited state of an electron also possesses certain polarisation features. When light polarisation is aligned along a given chemical axis, fluorophores are activated preferentially (the excitation dipole).

In addition, the succeeding fluorescence photons released by the molecule will have polarisation aligned along another molecular axis (the emission dipole). Excitation and emission dipoles do not typically overlap.

Operating Procedure of Fluorescence spectroscopy

This device consists of a light source, two monochromators, a sample holder, and a detector.
There are two monochromators, one for selecting the wavelength of stimulation and the other for analysing the emitted light.

The detector and the excitation beam are at right angles.
When sample molecules are excited, fluorescence is emitted in all directions and detected by a photocell at right angles to the excitation light beam.
The lamp source employed emits radiation in the ultraviolet, visible, and near-infrared spectrums.

An optical system directs the light to the excitation monochromator, which permits preselection or scanning of a certain wavelength range.
The stimulating light is subsequently sent to the sample compartment containing the fluorescence cuvette.
A fluorescent cuvette with translucent quartz or glass sides is utilised.

When excited light strikes the sample cell, molecules in the solution get excited and some emit light.
The emission monochromator examines light emitted perpendicular to the incoming beam.
The wavelength analysis of emitted light is performed by measuring the fluorescence intensity at a predetermined wavelength.

The analyzer monochromator directs the selected wavelength of emitted light to the detector.
A photomultiplier tube is the detector used to measure the light’s intensity.
The photomultiplier’s output current is sent to a measuring device that indicates the level of fluorescence.

Types of luminescence

By Mechanism

1. Fluorescence:

Fluorescence is a phenomenon in which a substance absorbs light at a specific wavelength, called the excitation wavelength, and then re-emits light at a longer wavelength, called the emission wavelength. This re-emitted light is called fluorescence.
The process of fluorescence occurs when an electron in a molecule is excited from its ground state to a higher energy state by absorbing a photon of light. Once the electron is in the excited state, it quickly relaxes back to the ground state, releasing energy in the form of a photon of light. This emitted light has a longer wavelength than the excitation light, which results in the characteristic shift in wavelength from excitation to emission.
Fluorescence is a property of many organic and inorganic molecules, including dyes, pigments, and biomolecules such as proteins and nucleic acids. Fluorescence can be used to study the properties of these molecules and to detect their presence in a sample.

Fluorescence spectroscopy is a technique that is based on the principle of fluorescence. It is used to measure the intensity of light emitted by a substance after it has been excited by a specific wavelength of light. This technique is used to study the properties of molecules and to detect the presence of specific substances in a sample.

2. Phosphorescence

Phosphorescence is a phenomenon that is similar to fluorescence, but with a longer-lived excited state. In phosphorescence, a substance absorbs light at a specific wavelength, called the excitation wavelength, and then re-emits light at a longer wavelength, called the emission wavelength. However, unlike fluorescence, the excited state in phosphorescence has a longer lifetime, which means that the emission can continue for a period of time even after the excitation light is turned off.
Phosphorescence is a result of a transition from a triplet state, which is a higher energy state than the singlet state. The excited electron in a triplet state can relax back to the ground state by emitting a photon of light, but it can also relax to the first excited state and from there it will emit a photon of light. This process of relaxation from the triplet state to the ground state is much slower than the process of relaxation from the singlet state to the ground state, which results in the longer lifetime of the excited state in phosphorescence.

Phosphorescence is observed in some organic and inorganic molecules, including some dyes, pigments, and biomolecules such as proteins and nucleic acids. Phosphorescence can be used to study the properties of these molecules and to detect their presence in a sample.
Phosphorescence spectroscopy is a technique that is based on the principle of phosphorescence. It is used to measure the intensity of light emitted by a substance after it has been excited by a specific wavelength of light. This technique is used to study the properties of molecules and to detect the presence of specific substances in a sample. However, it is not as widely used as fluorescence spectroscopy.

By Excitation Source:

1. Chemiluminescence

Chemiluminescence is the process through which light is produced as a result of a chemical reaction. Unlike fluorescence and phosphorescence, which entail a substance absorbing and re-emitting light, chemiluminescence involves the transfer of chemical energy into light energy.

A chemical reaction between two or more reactants results in the formation of one or more light-emitting species in chemiluminescence. These light-emitting entities could be excited atoms, ions, or molecules. They emit light as they return to their ground state.
The glow of fireflies is a well-known example of chemiluminescence. A chemical reaction between a substrate and an enzyme called luciferase produces light in fireflies.
Chemiluminescence is also employed in a variety of practical applications, including forensics, medical diagnostic assays, and environmental monitoring. It is employed, for example, in diagnostic assays for detecting bacterial infections as well as in the detection of chemical contaminants in water and air.

Chemiluminescence is a potent instrument for detecting low-concentration chemical species, and it has a high sensitivity and specificity for measuring chemical processes. It is also reasonably inexpensive and simple to use, making it accessible to a wide range of academics and professionals.

2. Cathodoluminescence

Cathodoluminescence (CL) is the phenomenon of a substance emitting light when exposed to a high-energy electron beam. It is a type of electron-induced luminescence in which a material is activated by a beam of high-energy electrons and produces light as a result.
CL is a surface-sensitive technique, which means it only detects light emitted from a sample’s top few microns. It is used to investigate the characteristics of many different materials, including semiconductors, minerals, ceramics, and polymers.

CL is utilised in a variety of applications, including the investigation of the electrical and optical properties of semiconductors and minerals, the composition and morphology of thin films, and the investigation of flaws and impurities in materials. It is also useful in geology, where it is used to investigate the composition and structure of minerals and rocks.
CL is a useful technique for material characterization, providing information regarding crystal structure, composition, and electrical characteristics. It is also reasonably inexpensive and simple to use, making it accessible to a wide range of academics and professionals.
However, the sample must be conductive and the electron beam must be able to reach the sample surface, therefore it is not suited for nonconductive samples or samples buried deep in other materials. Furthermore, CL is ineffective for examining samples that are unstable under high-energy electron irradiation.

3. Electroluminescence

The phenomenon of light emission by a substance when an electric current is conducted through it is known as electroluminescence (EL). It’s an example of electrically induced light. In EL, a material is stimulated by an electrical current and emits light as a result.
Light-emitting diode (LED) technology is the most prevalent type of EL, and it is widely utilised in electronic products such as televisions, smartphones, and traffic lights. As the light-emitting layer of LEDs, a semiconductor material is utilised, and a voltage is put across it to form an electrical current, which causes the material to emit light.
EL is also employed in various applications like as flat-panel displays, illumination, and the investigation of material properties. It is a potent instrument for material characterization, providing information about electronic characteristics and studying charge transport in materials.

Because EL is relatively inexpensive and simple to use, it is accessible to a wide range of researchers and scientists. Furthermore, EL is energy-efficient, long-lasting, and extremely versatile; it can be used to create a wide variety of colours and brightness levels, as well as for a wide variety of applications.
However, because it requires a power source to operate, EL is not suited for applications where a power source is not available. Furthermore, EL is not appropriate for samples that are not conductive or are not stable under high-voltage or high-current situations.

4. Photoluminescence

Photoluminescence (PL) is the phenomenon of a material emitting light when exposed to light. It is a type of light-induced luminescence in which a substance is activated by light and produces light as a result. The light that is emitted can be fluorescence or phosphorescence.

A substance absorbs a photon of light, which excites an electron from its ground state to a higher energy state in photoluminescence. After a while, the electron returns to its ground state, producing a photon of light. The emitted light has a greater wavelength than the excitation light, resulting in the typical wavelength shift from excitation to emission.
Many organic and inorganic compounds, including dyes, pigments, and biomolecules such as proteins and nucleic acids, exhibit photoluminescence. Photoluminescence can be used to investigate these molecules’ characteristics and detect their existence in a sample.
Photoluminescence spectroscopy is a technique based on the photoluminescence principle. It is used to quantify the amount of light emitted by a substance after being activated by a given wavelength of light. This approach is used to investigate molecular characteristics and detect the presence of specific chemicals in a sample.

PL is a surface-sensitive technique, which means it only detects light emitted from a sample’s top few microns. It is reasonably inexpensive and simple to use, making it accessible to a wide range of academics and professionals. However, the samples must be able to fluoresce or phosphoresce, which means it is not suited for samples lacking such capabilities.

What is a Fluorescence Spectrum?

The emission and excitation spectra for a given fluorophore are mirror images of each other

Steady state fluorescence spectra are generated when molecules activated by a steady source of light emit fluorescence and the released photons, or intensity, are measured as a function of wavelength. When the excitation wavelength is held constant and the emission wavelength is scanned to produce a plot of intensity versus emission wavelength, the result is a fluorescence emission spectrum.

When the emission wavelength is held constant and the excitation monochromator wavelength is scanned, the result is a fluorescence excitation spectrum. Thus, the spectrum provides information regarding the wavelengths at which a sample will absorb in order to emit at the single emission wavelength chosen for observation. In terms of detection limits and molecular specificity, this approach is significantly more sensitive than absorbance spectrum. Excitation spectra are unique to a single emitting wavelength/species, whereas absorbance spectra measure all absorbing species in a solution or material. The emission spectrum and excitation spectrum of a given fluorophore are mirror reflections of one another. In general, the emission spectrum has longer wavelengths (lower energy) than the excitation or absorption spectrum.

These two sorts of spectral data (emission and excitation) are utilised to determine how a sample is transforming. Variables such as temperature, concentration, or interactions with other molecules in the vicinity may alter the spectral intensity and/or peak wavelength. This comprises molecules that serve as a quencher as well as molecules or materials involved in energy transfer. Some fluorophores are also sensitive to solvent environment characteristics such as pH, polarity, and ion concentrations.

Characteristics of a Fluorescence Spectrum

Fluorometers consist of an excitation monochromator and an emission monochromator, enabling users to acquire both excitation and emission spectra. A fluorometer’s measurement is exclusive to the instrument’s excitation and emission monochromators. Fluorescence is directly proportional to luminous flux and measurement efficiency, and is thus dependent on instrument design and components such as the light source, monochromator optics, and photomultiplier tube. Each light source will have a unique spectral output (both shape and intensity) that will vary and diminish over its lifetime.

Excitation spectra depict the intensity at a constant emission wavelength while altering excitation wavelengths. Since the majority of emission spectra are independent of the excitation wavelength, the excitation spectrum of a fluorophore is frequently identical to its absorption spectrum.

Characteristics of a Fluorescence Spectrum — Cartoon schematic of a fluorometer. | Image Source: https://jascoinc.com/learning-center/theory/spectroscopy/fluorescence-spectroscopy/

In contrast, emission spectra plot the intensity at a constant excitation wavelength while scanning through various emission wavelengths. These emission scans provide information on the molecular structure and immediate surroundings of the fluorophore. Since fluorescence emission always occurs from the lowest excited state to the ground state, the excitation wavelength has no effect on the structure of the emission spectrum. Additionally, more energy is required to excite a molecule from its ground state to its excited state, resulting in emission peaks with longer wavelengths (i.e. lower energies) than their respective excitation wavelengths. The Stokes shift refers to this differential in energy between the excitation and emission wavelengths.

Due to the equal distribution of vibrational energy levels between the excited and ground states, absorption and emission spectra are commonly mirror images of one another. The Franck-Condon principle explains that because the nuclei are relatively large and the electronic transitions involved in emission and absorption occur on such short timescales, there is no time for the nuclei to move and the vibrational energy levels therefore remain relatively constant during the electronic transition.

Instrumentation of fluorescence spectroscopy

Major components of fluorescence spectrophotometry.

All fluorescence instruments include three fundamental components: a light source, a sample holder, and a detector. In addition, the wavelength of incident radiation must be selectable and the detector signal must be capable of precise adjustment and presentation for analytical purpose.

In basic filter fluorimeters, the wavelengths of stimulated and emitted light are chosen via filters, allowing for measurements at any pair of fixed wavelengths. Either a continuously changing interference filter or a monochromator can be used to analyse the spectral distribution of the light emitted from the sample, the fluorescence emission spectrum, in simple fluorescence spectrometers.

In more complex instruments, monochromators are used for both the selection of excitation light and the analysis of sample emission. These instruments can also measure the fluctuation of emission intensity with excitation wavelength, also known as the fluorescence excitation spectrum.

In theory, the greatest sensitivity can be attained by employing filters that permit the collection of the full spectrum of wavelengths emitted by the sample, in conjunction with the most intense source available. In fact, in order to exploit the full potential of the approach, only a narrow band of emitted wavelengths is evaluated, and the incident light intensity is not made excessive in order to limit the likelihood of sample photodecomposition.

Essential components of a fluorescence spectrometer

1. Light Sources for Fluorescence Spectroscopy

For fluorescence spectroscopy, various types of light sources can be utilised depending on the chosen method:

Continuous or pulsed emission can be utilised to generate UV light from simple light sources, such as some gas discharge lamps (e.g. mercury vapour lamps). Consequently, one frequently uses a bandpass optical filter to narrow the spectral spectrum. During measurements, there are also sources with an excitation monochromator that scan through a specified range.
In certain instances, even light-emitting diodes (LEDs) are adequate. This permits inexpensive and small solutions, especially for portable devices.

Different types of lasers are very potent excitation sources due to their ability to address a broad spectral range, including the ultraviolet, which is often generated by frequency doubling in a nonlinear crystal. Excitation with light with a narrow optical bandwidth may be advantageous (linewidth). In addition, with pulsed lasers, it is simple to generate nanosecond- or even picosecond- or femtosecond-long pulses.
For excitation with widely varied wavelengths, one can utilise tunable lasers, optical parametric oscillators (OPOs), or a broadband source in conjunction with an excitation monochromator, as stated previously. In the latter scenario, available excitation intensities are obviously significantly lower than with lasers.

Emission spectrum of xenon lamp used in the PerkinElmer LS Series

If the utilised light source cannot generate light with perfectly constant optical power or pulse energy, it is also possible to measure its output with an extra photodetector and use its output signal to eliminate power fluctuations from the fluorescence measurement findings.

2. Photodetectors

Depending on the requirements, different types of photodetectors might be employed. A photodetector with high sensitivity in the relevant spectral region, such as a photodiode, is all that is required for equipment with continuous-wave excitation and a scanning monochromator. In tandem with a dual monochromator for optimal suppression of stray light, an even more sensitive detector is required.
The usage of a spectrograph with a diffraction grating and a linear photodiode array or another type of photodetector with spatial resolution is another option. Using a two-dimensional sensor, such as an image sensor, it is possible to attain both one-dimensional spatial resolution and spectral resolution.
In addition to great sensitivity, fluorescence lifespan measurements require a quick response (high bandwidth). This reduces the number of acceptable detectors; photomultipliers and avalanche photodiodes are commonly used. Additionally, there are devices that contain a microchannel plate.

3. Wavelength selection

The simplest filter fluorimeters isolate both the stimulated and emission wavelengths using set filters. To isolate a specific wavelength from a source outputting a line spectrum, only two cut-off filters are necessary. These may be glass filters or solutions contained in cuvettes.
The emission filter must be selected so that Rayleigh-Tyndall scattered light is blocked and sample-emitted light is transmitted. In order to prevent high blanks, it may be required to filter away any Raman scatter. Recently, interference filters with high transmission ( ≈ 40%) of a small range (10 – 15 nm) of wavelengths have become commercially accessible, and filters with maximum transmission at any desired wavelength can be purchased.
However, these UV filters are pricey and have a restricted selection. For the majority of quantitative work, a basic filter system is suitable, especially when sufficient chemistry has been performed to eliminate interfering molecules. However, it is advantageous to be able to scan the sample’s emission to detect contaminants and improve conditions.

Utilizing a continuous interference filter to record an emission spectrum, at least in the visible section of the spectrum, is a practical method. Using monochromators to select both the excitation and emission wavelengths would be a further improvement. For this purpose, the majority of current instruments of this type utilise diffraction grating monochromators. This type of fluorescence spectrometer is capable of recording both excitation and emission spectra, maximising the technique’s analytical capability.
If monochromators are utilised, the slit width of both the excitation and emission monochromators should be adjustable independently. Numerous analyses will not necessitate high resolution (basically corresponding to high selectivity), and wider slit widths will result in increased sensitivity. To record the fine structure in the emission of, for instance, polyaromatic hydrocarbons or to selectively stimulate one component in the presence of another, however, small slit widths will be required, at the expense of sensitivity.

4. Read-out devices

The output from the detector is amplified and shown on a metre or digital display that serves as a readout device. It should be possible to adjust the sensitivity of the amplifier in discrete increments, allowing for the comparison of samples with vastly different concentrations. A continuous sensitivity adjustment is especially advantageous, as it enables the display to read immediately in concentration units.

Digital displays are the most legible and free of ambiguity. By employing integration techniques in which the average value over a few seconds is shown as a constant signal, precision can be increased. Microprocessor-based electronics produce outputs that are immediately compatible with printer systems and computers, thereby removing the chance of user mistake during data transfer.

5. Sample holders

Most fluorescence experiments are done in solution and measured in a cuvette or flowcell. Cuvettes can be circular, square, or rectangular (rarely rectangular) and made of a material that transmits incident and emitted light.
Since pathlength and parallelism are easier to maintain throughout fabrication, square cuvettes or cells are most exact. Round cuvettes are cheaper and ideal for many routine uses. The cuvette faces the beam.

The fluorescence is evenly distributed and can be collected from the cell’s front surface, at right angles to the incident beam, or in-line with it. Some equipment allow sample attributes to determine the collecting method. A very dilute solution will glow evenly along the incident beam’s course through the sample.
The right-angled collecting approach minimises light scattering by the solution and cell under these conditions. Analytical measurements use this circumstance. Only a small fraction of fluorescence from every point along the light path is caught by the device and sent to the detector.
The upshot is that much of the solution does not contribute to fluorescence emission and the same intensity will be recorded from a significantly smaller volume of solution contained in a microcell whose dimensions better meet the instrument’s optical considerations.

As the solution absorbs more, the fluorescence emission becomes distorted until it only penetrates the cuvette’s front surface. Front surface collection will allow measurements, however light dispersed from the cuvette wall will be significant.
Front surface collection will always display emission (perhaps distorted) from a fluorescent sample, while 90° collection fluorescence gradually decreases as solution absorbance increases. High concentration can make a sample non-fluorescent. Before doing a fluorescence check, measure the absorbance of a fully unknown solution and adjust the concentration to <0.1 A.

Fluorescence emission from a microcell whose dimensions closely match the optical considerations of the instrument

Factors that affect fluorescence spectroscopy

Molecular rigidity: For fluorescence spectroscopy, rigid fluorophores are chosen since they have less vibrations and a lower probability of changing to triplet state. Fluorescein and eosin have hard structures and are very luminous, but phenolphthalein has a flexible structure and is not fluorescent.

Solvent polarity: The degree of fluorescence can also be determined by the polarity of the solution. In the presence of heavy atoms in the solvent, the fluorescence of the structures can diminish.
Dissolved oxygen: Oxygen dissolved in the solvent can also reduce the intensity of fluorescence emission. Fluorophore undergoes photochemical oxidation to accomplish this. Oxygen’s paramagnetic characteristics can also result in fluorescence quenching.
pH: pH can impact the fluorescence of a substance. An illustration of this is aniline, which is a cation at low pH and an anion at high pH. In both situations, fluorescence is lost.

Quenching: Quenching refers to a decrease in the intensity of fluorescence. This may be the result of fluorescence being absorbed by the solution or the fluorescent substance itself being absorbed. This effect is known as self-quenching.
Conjugation: Molecules must be unsaturated, i.e., they must have π electrons, in order to absorb UV/vis radiation. If there is no absorption of radiation, fluorescence will not occur.
Rigidity of structures: Rigid structures will emit more fluorescence, whereas flexible structures would emit less.

Nature of substituent groups: Electron-donating groups, such as amino and hydroxyl, boost fluorescence activity. Electron-withdrawing groups, such as Nitro and carboxyl, decrease fluorescence. Fluorescence intensity is unaffected by groups such as SO3H and NH4+.
Effect of temperature: Increase in temperature causes an increase in molecular collisions and a fall in fluorescence intensity, whereas a decrease in temperature causes a decrease in collisions and an increase in fluorescence intensity.
Viscosity: Increased viscosity decreases molecular collisions, which increases fluorescence intensity, whereas decreasing viscosity increases molecular collisions, which decreases fluorescence intensity.

Applications of fluorescence spectroscopy

Applications of fluorescence spectroscopy are nearly limited only by the imagination. These days, it is difficult to envision the chemical and biological sciences without this approach.

In the summary that follows, only chosen instances of application per industry for some of the most prevalent industries in which it is employed will be provided.

1. Bioscience

One of the most common applications of fluorescence spectroscopy in biosciences is the precise measurement of DNA and RNA. A DNA sample is treated with an extrinsic fluorophore (typically ethidium bromide) and put into a fluorescence spectrometer to determine the sample’s concentration.

SMRT (single-molecule real-time) DNA sequencing is a further current use. It is anticipated to be essential to the next revolution in genetic diagnostics due to its capacity to manufacture long-read single molecules with great precision.

2. Industrial

In a number of industrial situations, fluorescence spectroscopy is utilised as a rapid, non-invasive tool for assessing pollution. It has been used, for instance, to detect contaminating organic chemicals in groundwater following hydraulic fracturing for gas exploration.

3. Chemical

In the realm of nanoparticle production for possible medicinal applications, such as drug delivery, fluorescence spectroscopy has a significant chemical use.

When nanoparticles are exposed to biological fluids, proteins and other biomolecules coat them (called the protein corona). The interactions between the nanoparticle and the protein corona have consequences for the safe administration of the nanoparticle in vivo.
combine time-resolved fluorescence quenching and fluorescence correlation spectroscopy to explore these interactions and gain a deeper understanding of nanotechnology.

4. Environmental

The approach also has a broad applicability in environmental monitoring. The remediation of water surrounding landfills is one example.

Landfill leachates form as rainwater percolates through garbage and liquid forms during biodegradation of waste in a landfill. Pollutants are present in leachate, which can be hazardous to the environment.
Leenheer and Croué (2003) and Zhang et al. (2013) use high-resolution fluorescence spectroscopy and 3D-excitation emission matrix fluorescence spectroscopy to analyse dissolved organic matter in these samples and optimise treatment techniques for landfill leachate, respectively.

5. Pharmaceutical

In the pharmaceutical industry, spectrofluorometric techniques are also utilised to analyse pharmaceuticals. The study of co-formulated pills provided as cholesterol treatment is one example.

Synchronous fluorescence spectroscopy is a straightforward, rapid, and precise method for testing the tablet Atoreza, which contains both Ezetimibe and Atorvastatin calcium. This technique is appropriate for routine quality control of this drug.

6. Agricultural

Spectroscopic techniques are also commonly used in agriculture, for instance in the identification of various crop kinds. LIFS (laser-induced fluorescence emission technique) is a good method for identifying citrus seedling varieties.
Similarly, tea businesses can use total luminescence spectroscopy as a rapid, economical, and objective alternative to engaging professional tea tasters to distinguish between comparable types of tea.

Due to its nearly 70-year history, fluorescence spectrophotometry has developed into a method with several complex applications.
Any specific context in which it can be employed necessitates extensive investigation into the optimal application strategy to get the desired results.
Scientists desiring a deeper understanding of fluorescence spectrophotometry have access to a plethora of scholarly resources and commercial solutions.

7. Other Applications

Fluorescence spectroscopy is a versatile technique that is used in a wide variety of applications. Some of the main applications include:

Biochemistry and molecular biology: Fluorescence spectroscopy is used to study the structure and function of biomolecules such as proteins, nucleic acids, and lipids. It is also used to study the interactions between these molecules and other molecules, such as drugs and small molecules.
Medical diagnostics: Fluorescence spectroscopy is used to detect the presence of disease markers in biological samples such as blood, urine, and tissue samples. It is also used in imaging techniques such as fluorescence microscopy to study the distribution of disease markers in tissue samples.

Environmental monitoring: Fluorescence spectroscopy is used to monitor the presence of pollutants and other contaminants in water, air, and soil samples.
Food safety and quality control: Fluorescence spectroscopy is used to detect the presence of contaminants and to monitor the quality of food products.
Industrial applications: Fluorescence spectroscopy is used in a wide variety of industrial applications, including quality control of industrial products, process monitoring, and the detection of impurities in raw materials.

Drug discovery and development: Fluorescence spectroscopy is used to study the interactions between drugs and biomolecules and to monitor the efficacy of drugs in vivo.
Nanotechnology: Fluorescence spectroscopy is used to study the properties of nanomaterials and to monitor the behavior of nanoparticles in biological systems.
Forensic science: Fluorescence spectroscopy is used to detect and identify traces of evidence in forensic investigations.

Overall, fluorescence spectroscopy is a powerful technique that provides valuable information in a wide range of applications.

Advantages of fluorescence spectroscopy

Fluorescence spectroscopy has several advantages over other spectroscopic techniques, some of them include:

High sensitivity: Fluorescence spectroscopy is able to detect very low concentrations of molecules with high sensitivity, making it an ideal tool for detecting trace amounts of biological molecules, pollutants, and other contaminants.

Specificity: Fluorescence spectroscopy can be used to selectively detect specific molecules or groups of molecules by using appropriate excitation and emission wavelengths.
Low background noise: Fluorescence spectroscopy generally has a low level of background noise, which makes it easy to detect weak fluorescence signals.
Multiplexing: Fluorescence spectroscopy can be used to detect multiple molecules simultaneously by using different fluorescent labels.
Non-destructive: Fluorescence spectroscopy can be used to study samples without altering or destroying them.
In-vivo imaging: Fluorescence spectroscopy can be used in-vivo with the help of fluorescent labels which can be targeted to specific cells or tissues and can be used to track the movement of biomolecules in living organisms.
Cost-effective: Fluorescence spectroscopy equipment is relatively inexpensive and easy to use, making it accessible to many researchers and scientists.
Versatility: Fluorescence spectroscopy can be used in a wide variety of applications, including chemistry, biology, medicine, environmental monitoring, and industrial analysis.

Overall, fluorescence spectroscopy is a powerful and versatile tool that can be used to study a wide range of samples and provide valuable information in a wide range of applications.

Disadvantages of fluorescence spectroscopy

While fluorescence spectroscopy has many advantages, it also has some limitations and disadvantages. Some of the main disadvantages include:

Photobleaching: Fluorescence spectroscopy can cause the fluorescence signal to decrease over time due to photobleaching, which occurs when the fluorescent molecules are exposed to light and lose their fluorescence.
Quenching: Some molecules can quench the fluorescence of other molecules, leading to a decrease in the fluorescence signal.
Interference from other sources: Fluorescence spectroscopy can be affected by interference from other sources such as scatter, absorption, and autofluorescence, which can make it difficult to interpret the results.
Limited penetration depth: Fluorescence spectroscopy is limited by the penetration depth of the excitation light, which means that it is not suitable for studying deep tissue samples or samples that are not transparent to the excitation light.
Fluorophore dependent: The fluorescence signal is dependent on the presence of fluorescent molecules, so it is not suitable for samples that do not contain fluorescent molecules.
Limited to certain molecular species: Fluorescence spectroscopy can only be used to detect molecules that can fluoresce, which means that it is not suitable for detecting all types of molecules.
Limited dynamic range: Fluorescence spectroscopy has a limited dynamic range, meaning it can only detect a certain range of concentration and not very high or very low.
High-cost of reagents: Some applications of fluorescence spectroscopy require the use of expensive reagents, such as fluorescent labels.
Others
- As fluorescence intensity may be highly reliant on buffering, it is vital to buffer with care.
- Excitation with ultraviolet light may result in photochemical changes or the destruction of the fluorescent molecule.
- Increased photochemical damage may result from the presence of dissolved oxygen.
- Trace amounts of iodide and nitrogen oxides are effective quenchers, and as such, they interfere.
- The approach is unsuitable for determining a sample’s principal constituents due to its low accuracy for big volumes.
- Because not every element or compound may glow, the scope of this technique’s applicability is limited.

Overall, while fluorescence spectroscopy is a powerful and versatile tool, it also has limitations and disadvantages that should be considered when planning experiments and interpreting results.

Precautions

Fluorescence analysis is particularly appropriate to trace chemicals; nevertheless, sample contaminations must be eliminated with care. Rubber and cork stoppers contain fluorescent pigments, which are removed when they come into contact with a solvent.
Additionally, fluorescent material is removed from filter paper using solvents.
Fluorescent contaminants include grease from stop co**s and other sources.
All glasses contain the extractable elements Al, Ca, and SiO2.
The most significant aspect is the reagent concentration. Concentration must be stated in micromoles so that the reagent-to-metal ratio can be easily determined. Large differences in temperature between the unknown and standard should be avoided.
Additionally, prolonged exposure of the solution to ultraviolet radiation is undesirable.

FAQ

What is Fluorescence Spectrophotometry?