12.7
Spectromètres UV-Visible
L'absorption des rayonnements UV et visibles (UV-visible) est mesurée à l'aide d'un spectrophotomètre UV-visible. Les lampes au deutérium, qui émetten…
Un spectromètre UV-visible se compose d’une source lumineuse, telle qu’une lampe au deutérium pour la région UV et une lampe au tungstène pour la région visible, un monochromateur, un porte-échantillon et un détecteur.
La spectroscopie visible utilise des cellules d’échantillon de verre ou de plastique, tandis que la spectroscopie UV utilise des cellules de quartz.
Le prisme divise le faisceau de lumière en ses longueurs d’onde composantes, et un système de fentes et de filtres concentre la longueur d’onde souhaitée sur la cellule d’échantillon.
Le détecteur à photodiodes mesure l’intensité de la lumière traversant la cellule d’échantillon.
Dans les instruments à faisceau unique, le monochromateur placé entre la source et l’échantillon permet l’analyse d’une longueur d’onde à la fois.
Alternativement, dans un instrument à double faisceau, le monochromateur est équipé de séparateurs et de miroirs, qui divisent la lumière en deux faisceaux. Le faisceau d’échantillon passe à travers la cellule d’échantillon et le faisceau de référence passe à travers l’échantillon de référence.
Les deux spectres sont soustraits pour retirer les contributions de l’échantillon de référence du spectre brut acquis.
View the full transcript and gain access to JoVE Core videos
Q1: What are the main components of a UV-Vis spectrometer?
A UV-Vis spectrometer contains a light source (deuterium lamp for UV, tungsten lamp for visible light), a monochromator to split light into component wavelengths, a sample holder or cuvette, and a detector such as a photodiode. These components work together to measure how much light a sample absorbs at specific wavelengths during molecular electronic transitions.
Q2: Why are different cuvette materials used for UV versus visible spectroscopy?
Glass and plastic cuvettes are used for visible spectroscopy because they effectively transmit visible light with spectral cutoffs around 350 nm. UV spectroscopy requires quartz cuvettes with lower cutoffs around 200 nm to allow transmission of UV radiation. This material selection ensures accurate absorbance measurements across different wavelength regions.
Q3: How does a monochromator function in UV-Vis spectrometers?
A monochromator uses a prism or diffraction grating to split incoming light into its component wavelengths. A system of slits and filters then focuses the desired wavelength onto the sample cell. This allows the spectrometer to analyze one specific wavelength at a time, enabling precise measurement of light absorption.
Q4: What is the difference between single-beam and double-beam instruments?
In single-beam instruments, all light passes through the sample cell sequentially. Double-beam instruments split light into two beams using mirrors and splitters: one passes through the sample, the other through a reference. The spectra are subtracted to remove reference contributions, providing more accurate measurements by compensating for instrumental variations.
Q5: Why is solvent selection critical in UV-Vis spectroscopy?
Solvents absorb light and affect spectral fine structure. Nonpolar solvents like hexane do not form hydrogen bonds with solutes, preserving fine structure similar to gaseous phase spectra. Polar solvents like water form hydrogen bonds, eliminating fine structure. Choosing an appropriate solvent ensures accurate absorbance measurements and reveals desired spectral details.
Q6: What role does the detector play in measuring light intensity?
Detectors such as photodiodes, photomultiplier tubes, and photodiode arrays measure the intensity of light passing through the sample cell, referred to as I. Modern diode-array spectrophotometers use photodiode detectors to record the entire spectrum simultaneously, enabling rapid and comprehensive analysis of sample absorbance across multiple wavelengths.
Q7: Why must samples be in liquid form for UV-Vis spectrophotometry?
UV-Vis spectrophotometers require samples in liquid phase for accurate analysis. Solid organic compounds must be dissolved in a suitable solvent prior to measurement. This ensures uniform light interaction with the sample and allows the spectrometer to measure absorbance reliably. Understanding interaction of EM radiation with matter spectroscopy principles guides this requirement.