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UV-Vis Molecular Absorbance Spectroscopy


Light Through SampleIn molecular absorbance spectroscopy, a beam of ultraviolet or visible light is directed through a sample. Some of the light may be transmitted through the sample. Light that was not transmitted through the sample was absorbed.
Transmittance (T) is defined as the ratio of P/Po.
Absorbance (A) is defined as -log(P/Po).

AbsorbanceA molecule can absorb some of the light only if it can accommodate that additional energy by promoting electrons to higher energy levels. The energy of the light being absorbed must match the energy required to promote the electron. Therefore, not all wavelengths of light are absorbed equally by a sample. An absorbance spectrum depicts what wavelengths of light are absorbed by a sample. The UV-Vis absorbance spectrum below was obtained by passing different wavelengths of light through a solution of fabric dye, and measuring the intensity of light (P) passing through the solution. One can readily see what wavelengths of light are absorbed (peaks), and what wavelengths of light are transmitted (troughs).
Absorbance Spectrum

One very important relationship in absorbance spectroscopy is Beer's Law:
where: A is absorbance
a describes the ability of a molecule to absorb radiation at a particular wavelength.
b is the length of sample through which the light beam passes.
c is the concentration of the absorbing species.

This relationship is the basis of all quantitative work in absorbance spectroscopy. It allows one to determine the concentration of an absorbing species simply by measuring its absorbance.

KU Spectrophotometers

Varian Cary 50

Varian Cary 50The Cary 50 design is simple, yet elegant. Its fast-scanning monochromator collects spectra in seconds, and the Xe flash lamp is pulsed to avoid interference from room light. The Cary 50 is controlled by intuitive software which allows students who are unfamiliar with spectrophotometers to begin collecting data without extensive software training.

We have purchased several accessories for use with this instrument, which include a peltier thermostatted cell holder to rapidly change sample temperature between 0 to 100 degrees Celsius, sample temperature probes to accurately determine the temperature of the sample inside the cuvette, and a stopped flow (rapid kinetics) accessory to measure kinetics of fast reactions (of less than a second).

BioTek Synergy2BioTek Synergy2 Plate Reader

The Synergy 2 Plate Reader is capable of measuring absorbance or fluorescence of many samples in a fast, automated fashion that requires very little sample. The open drawer seen in the image of the instrument accommodates a well plate. We typically use plates with 96 separate compartments, so we can analyze up to 96 samples at a time.

Genesys 10 UV SpectrophotometersGenesys 10 UV Spectrophotometer

We have a fleet of 8 Genesys 10 UV spectrophotometers that are used in a variety of laboratory courses throughout the chemistry curriculum. These UV-Vis spectrophotometers are rugged, user-friendly, have a small footprint, and are great for quantitative work and kinetics at a single wavelength.

Hewlett Packard 8453 Diode ArrayHewlett Packard 8453 Diode Array

Diode array spectrometers contain thousands of individual photodetectors enabling them to measure the intensity at many wavelengths simultaneously. Absorbance spectra can be obtained in a fraction of a second, and several wavelengths of light can be monitored simultaneously during kinetic experiments.

Ocean Optics Miniature SpectrophotometersOcean Optics Miniature Spectrophotometer

Ocean Optics has taken the lead in miniaturizing diode array spectrometers. By using the diode array approach, there are no moving parts in the spectrometer. Fiber optic cables are used to take light from the source to the sample, and from the sample to the detector. These are versatile instruments that can be configured to apply to a variety of sampling situations.

Spectronic 20 Photometers

Spec 20s have been around since the 1960s, and we have a few of them. These spectrometers are rugged, rudimentary, reasonably priced, visible spectrometers. It is because of these qualities that it is not surprising to see Spec 20s still in use in many educational and industrial labs. The 8 Spec 20s we have are all operational. One of them has undergone a solid-state upgrade, and is fitted with a digital display. We have a box of spare vacuum tubes, light bulbs, and detectors to keep them going well into the next decade.
Spectronic 20 Photometer

A Few Applications of UV-Vis Molecular Absorbance Spectroscopy

Quantitative analysis of all molecules that absorb light
Disruption of DNA double helix
Molecular temperature probes
Determination of equilibrium constants
Enzyme Kinetics
Analysis of mixtures of absorbing species
UV-VIS absorption as a diagnostic for NO in rocket plumes
Analysis of foods
Monitor ozone depletion
Determination of micelle structure
Drug dissolution
Spectral analysis of paintings
Art restoration / color matching
Light harvesting for solar energy
Determining wavelength response of fish eyes - Absorption spectra of retinal pigments