12.7
UV-Vis-spectrometers
De absorptie van UV- en zichtbare (UV-zichtbare) straling wordt gemeten met een UV-zichtbare spectrofotometer. Deuteriumlampen, die UV-straling uitzen…
A UV–Visible spectrometer consists of a light source, such as a deuterium lamp for the UV region and a tungsten lamp for the visible region, a monochromator, a sample holder, and a detector.
Visible spectroscopy uses sample cells of glass or plastic, while UV spectroscopy uses cells of quartz.
The prism splits the beam of light into its component wavelengths, and a system of slits and filters focuses the desired wavelength on the sample cell.
The photodiode detector measures the intensity of light passing through the sample cell.
In single beam instruments, the monochromator placed between the source and the sample allows analysis of one wavelength at a time.
Alternatively, in a double beam instrument the monochromator is equipped with splitters and mirrors, which split the light into two beams. The sample beam passes through the sample cell, and the reference beam passes through the reference sample.
The two spectra are subtracted to remove contributions from the reference sample from the acquired raw spectrum.
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.