Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation, re-emitting it at a different, typically longer, wavelength. This process is a type of luminescence, producing light without heat, unlike incandescence. The phenomenon is temporary, ceasing almost immediately once the excitation light source is removed.
How a Fluorescence Spectrum is Formed
The formation of a fluorescence spectrum begins with the absorption of light by a molecule, known as excitation. When a photon strikes a molecule, its energy is absorbed by an electron, causing it to jump from the ground state to an excited state. This absorption process occurs within femtoseconds (10^-15 seconds).
Once in the excited state, the molecule is unstable. Electrons lose vibrational energy to the surrounding environment, typically within picoseconds (10^-12 seconds), through vibrational relaxation. This relaxation brings the electron to the lowest vibrational level of the first excited singlet state.
From this lowest excited singlet state, the electron transitions back to the ground state, emitting a photon. The emitted photon has less energy than the absorbed photon because some energy was lost as heat during vibrational relaxation, which results in the emitted light having a longer wavelength than the absorbed light.
The collection of emitted photons across different wavelengths, plotted against their intensity, forms the fluorescence emission spectrum. The excitation spectrum, conversely, plots the intensity of emitted fluorescence at a fixed emission wavelength as the excitation wavelength is varied. This spectrum often mirrors the molecule’s absorption spectrum, showing which wavelengths are most effective at exciting the molecule to fluoresce.
Characteristics of the Spectrum
A fluorescence spectrum is defined by several features, including its peak wavelength and intensity. The peak wavelength represents the wavelength at which the fluorescence emission is strongest. Intensity indicates the amount of light emitted, directly related to the concentration of the fluorescent substance.
The Stokes shift is the difference in wavelength between the peak of the excitation spectrum and the peak of the emission spectrum. This shift occurs because some energy is lost as heat through molecular vibrations and interactions with the solvent during the brief time the molecule is in its excited state. The larger the Stokes shift, the easier it is to distinguish the emitted fluorescence from the exciting light.
Environmental factors can influence the shape and intensity of a fluorescence spectrum. For example, solvent polarity can alter the Stokes shift; more polar solvents often lead to larger shifts due to greater stabilization of the excited state. Temperature also plays a role, as increasing temperature generally decreases fluorescence intensity by promoting non-radiative energy loss.
The pH of the solution and the presence of certain ions can also affect the spectral properties of a fluorophore. The concentration of the fluorescent substance also directly impacts the intensity of the emitted light; at low concentrations, fluorescence intensity is typically proportional to the substance’s concentration, forming the basis for quantitative analysis.
Practical Uses of Fluorescence Spectroscopy
Fluorescence spectroscopy is a versatile analytical technique with widespread applications across scientific and industrial fields due to its high sensitivity and specificity. In biological sciences, it is used for tracking molecules within living cells, allowing researchers to observe dynamic cellular processes. It also plays a role in advanced DNA sequencing methods, such as single-molecule real-time (SMRT) DNA sequencing, and in studies related to protein folding and interactions.
Environmental monitoring benefits from fluorescence spectroscopy, particularly in detecting pollutants. It helps characterize dissolved organic matter in landfill leachates and identify organic compounds in groundwater, especially after activities like hydraulic fracturing. This assists in assessing and managing environmental contamination.
In medical diagnostics, fluorescence spectroscopy is employed for identifying diseases and imaging tissues. It can detect bacteria, viruses, fungi, and parasites with high sensitivity. For instance, in cancer diagnostics, substances that preferentially accumulate in tumors can be detected using laser-induced fluorescence, aiding in early detection and treatment.
Materials science utilizes this technique for characterizing new materials and ensuring quality control. It is applied in the development and testing of semiconductor devices, optical devices, and materials like organic light-emitting diodes (OLEDs) and quantum dots. This allows scientists to analyze the properties and behavior of these materials.
Forensic science also relies on fluorescence spectroscopy for analyzing evidence. It is used to detect latent fingerprints, characterize inks in questioned documents, and analyze other types of evidence such as drugs, glass fragments, and biological samples. This technique provides precise measurements valuable for criminal investigations.