0:00
Overview
1:11
Principles of UV-Vis Spectroscopy
3:46
Absorbance Measurements with UV-Vis Spectroscopy
5:30
Kinetics Experiments with UV-Vis Spectroscopy
6:47
Applications
8:57
Summary
Ultraviolet-Visible (UV-Vis) Spectroscopy
Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Ultraviolet-visible (UV-Vis) spectroscopy is one of the most popular analytical techniques because it is very versatile and able to detect nearly every molecule. With UV-Vis spectroscopy, the UV-Vis light is passed through a sample and the transmittance of light by a sample is measured. From the transmittance (T), the absorbance can be calculated as A=-log (T). An absorbance spectrum is obtained that shows the absorbance of a compound at different wavelengths. The amount of absorbance at any wavelength is due to the chemical structure of the molecule.
UV-Vis can be used in a qualitative manner, to identify functional groups or confirm the identity of a compound by matching the absorbance spectrum. It can also be used in a quantitative manner, as concentration of the analyte is related to the absorbance using Beer's Law. UV-Vis spectroscopy is used to quantify the amount of DNA or protein in a sample, for water analysis, and as a detector for many types of chromatography. Kinetics of chemical reactions are also measured with UV-Vis spectroscopy by taking repeated UV-Vis measurements over time. UV-Vis measurements are generally taken with a spectrophotometer. UV-Vis is also a very popular detector for other analytical techniques, such as chromatography, because it can detect many compounds.
Typically, UV-Vis is not the most sensitive spectroscopy technique, because not a lot of light is absorbed over a short path length. Other spectroscopy techniques such as fluorescence have higher sensitivity, but they are not as generally applicable, as most molecules are not fluorescent. UV-Vis has a similar sensitivity to other absorbance measurements, such as infrared spectroscopy.
1. Calibrate the Spectrometer
- Turn on the UV-Vis spectrometer and allow the lamps to warm up for an appropriate period of time (around 20 min) to stabilize them.
- Fill a cuvette with the solvent for the sample and make sure the outside is clean. This will serve as a blank and help account for light losses due to scattering or absorption by the solvent.
- Place the cuvette in the spectrometer. Make sure to align the cuvette properly, as often the cuvette has two sides, which are meant for hand
UV-Vis can be used to obtain a spectrum of colored compounds. In Figure 1A, the absorbance spectrum of a blue dye is shown. The background shows the colors of light in the visible spectrum. The blue dye has a λmax absorbance in the orange/red. Figure 1B shows a spectrum of a red dye, with λmax in the green.
Kinetics can be measured from a plot of absorbance at one wavelength over time. Fi
UV-Vis is used in many chemical analyses. It is used to quantitate the amount of protein in a solution, as most proteins absorb strongly at 280 nm. Figure 3 shows an example spectra of cytochrome C, which has a high absorbance at 280 and also at 450 because of a heme group. UV-Vis is also used as a standard technique to quantify the amount of DNA in a sample, as all the bases absorb strongly at 260 nm. RNA and proteins also absorb at 260 nm, so absorbance at other wavelengths can be measured to check for
Tags
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved