Name: Ultraviolet-Visible (UV-Vis) Spectroscopy | Chemistry | Concepts, Methods & Applications | JoVE Science Education
Uploaded: 2023-04-30T06:45:54.000Z
Description: Watch how Ultraviolet-Visible (UV-Vis) Spectroscopy works through a 9-min animated science video. Part of the Chemistry - Analytical Chemistry collection on JoVE Science Education.

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1. Calibrate the Spectrometer
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Place the cuvette in the spectrometer. Make sure to align the cuvette properly, as often the cuvette has two sides, which are meant for handling (may be grooved) and are not meant to shine light through.</li>
	<li>Take a reading for the blank. The absorbance should be minimal, but any absorbance should be subtracted out from future samples. Some instruments might store the blank data and perform the subtraction automatically.</li>
</ol>
2. Perform an Absorbance Spectrum
<ol>
	<li>Fill the cuvette with the sample. To make sure the transfer is quantitative, rinse the cuvette twice with the sample and then fill it about &#190; full. Make sure the outside is clean of any fingerprints, etc.</li>
	<li>Place the cuvette in the spectrometer in the correct direction.</li>
	<li>Cover the cuvette to prevent any ambient light.</li>
	<li>Collect an absorbance spectrum by allowing the instrument to scan through different wavelengths and collect the absorbance. The wavelength range can be set with information about the specific sample, but a range of 200&#8211;800 nm is standard. A diode-array instrument is able to collect an entire absorbance spectrum in one run.</li>
	<li>From the collected absorbance spectrum, determine the absorbance maximum (&#955;max). Repeat the collection of spectra to get an estimate of error in &#955;max.</li>
	<li>To make a calibration curve, collect the UV-Vis spectrum of a variety of different concentration samples. Spectrometers are often limited in linear range and will not be able to measure an absorbance value greater than 1.5. If the absorbance values for the sample are outside the instrument&#39;s linear range, dilute the sample to get the values within the linear range.</li>
</ol>
3. Kinetics Experiments with UV-Vis Spectroscopy
<ol>
	<li>UV-Vis can be used for kinetics experiments by examining the change in absorbance over time. For a kinetics experiment, take an initial reading of the sample.</li>
	<li>Quickly add the reagent to start the chemical reaction.</li>
	<li>Stir it well&#160;to mix with the sample. If a small amount is added, this could be done in a cuvette. Alternatively, mix the reagent with sample and quickly pour some in a cuvette for a measurement.</li>
	<li>Measure the absorbance at the &#955;max for the analyte of interest over time. If using up the reagent being measuring (i.e. absorbance is going up because there is less reagent to absorb), then the decay will indicate the order of the reaction.</li>
	<li>Using a calibration curve, make a plot of analyte concentration vs time, converting the absorbance value into concentration. From there, this graph can be fit with appropriate equations to determine the reaction rate constants.</li>
</ol>

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...

ultraviolet-visible (uv-vis) spectroscopy

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&#160;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&#39;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.

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. Figure 2</st...

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Ultraviolet-visible (UV-Vis) spectroscopy is one of the most popular analytical ...

Ultraviolet-visible, or UV-Vis, spectroscopy is one of the most popular analytical techniques in the laboratory.In UV-Vis spectroscopy, light is passed through a sample at a specific wavelength in the UV?or visible spectrum. If the sample absorbs some of the light, not all of the light will be pass through, or be transmitted. Transmission is the ratio of the intensity of the transmitted light to the incident light, and is correlated to absorbance. The absorbance can be used in a quantitative manner, to obtain the concentration of a sample. It can also be used in a qualitative manner, to identify a compound by matching the measured absorbance over a range of wavelengths, called the absorbance spectrum, to the published data. This video will introduce UV-Vis spectroscopy, and demonstrate its use in the laboratory in determining sample concentration and reaction kinetics.When a photon hits a molecule and is absorbed, the molecule is promoted from its ground state into a higher energy state. The energy difference between the two is the band gap. The energy of the photon must exactly match the band gap in order for the photon to be absorbed. The chemical structure determines the band ga

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Practice quiz on UV-Vis spectroscopy and spectrophotometry. Test your knowledge of absorbance, transmission, Beer’s law, blank measurements, cuvettes, monochromators, and absorbance spectra. Review how UV-Vis is used to find sample concentration, track reaction kinetics, and measure proteins, bacterial cells, and NOx.

What does UV-Vis spectroscopy primarily measure?

Transmission is the ratio of transmitted light intensity to incident light intensity.

The energy difference between the ground state and a higher energy state is called the {{slot:blank_1}}.

Which expression best represents Beer’s law?

A device that separates wavelengths of light spatially is called a {{slot:dropdown_1}}.

What wavelength range corresponds to visible light in this context?

The blank is prepared using the sample solvent and its measurement is subtracted from the sample measurement.

To quantify an unknown sample, a {{slot:blank_1}} can be prepared using known analyte concentrations.

Why is absorbance measured over time in the blue dye and bleach experiment?

Which set lists only applications of UV-Vis spectroscopy mentioned here?

Ultraviolet-Visible (UV-Vis) Spectroscopy Quiz

This assessment quiz covers core UV-Vis spectroscopy concepts, including the condition for photon absorption based on molecular band gaps, the role of the monochromator in selecting wavelengths, qualitative compound identification from absorbance spectra, concentration determination using calibration curves when molar absorptivity is unknown, and proper cuvette cleaning to avoid measurement errors.

Video Quiz

This assessment quiz covers sample preparation and solvent selection, concentration determination, volumetric glassware, internal standards, standard addition, and calibration curves. It also assesses core principles and practice in UV-Vis, Raman, XRF, GC-FID, and HPLC, including detection limits, matrix effects, scan resolution, retention behavior, and separations.

Chapter Quiz

Chemistry - Analytical Chemistry

Watch how Ultraviolet-Visible (UV-Vis) Spectroscopy works through a 9-min animated science video. Part of the Chemistry - Analytical Chemistry collection on JoVE Science Education.

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