UV-VIS Spectroscopy

I. Introduction

Electromagnetic Spectrum

For convenience in talking about electromagnetic radiation, we classify photons of different energies into different spectral regions. The photons in all of these regions have the same electromagnetic nature, but because of their very different energies they interact with matter very differently. For example, the human eye can only detect radiation that is in the visible region of the spectrum (hence the name). These photons are both transmitted by the lens of the human eye and absorbed by the photoreceptors in the retina. There is no fundamental difference in the nature of electromagnetic radiation of 350 nm versus 400 nm, other than our eyes can sense the 400-nm photons directly. A 350-nm photon is in the ultraviolet portion of the electromagnetic spectrum. Some of the boundaries between spectral regions are not well-defined as between ultraviolet and visible radiation.

Visible Spectrum

The visible region of the electromagnetic spectrum consists of photons with wavelengths from approximately 400 to 700 nm. The short wavelength cutoff is due to absorption by the lens of the eye and the long wavelength cutoff is due to the decrease in sensitivity of the photoreceptors in the retina for longer wavelengths. Light at wavelengths longer than 700 nm can be seen if the light source is intense.

II. UV-VIS Spectroscopy

Ultraviolet and visible (UV-Vis) absorption spectroscopy is the measurement of the attenuation of a beam of light after it passes through a sample or after reflection from a sample surface. Absorption measurements can be at a single wavelength or over an extended spectral range. Ultraviolet and visible light are energetic enough to promote outer electrons to higher energy levels, and UV-Vis spectroscopy is usually applied to molecules or inorganic complexes in solution. The UV-Vis spectra have broad features that are of limited use for sample identification but are very useful for quantitative measurements. The concentration of an analyte in solution can be determined by measuring the absorbance at some wavelength and applying the Beer-Lambert Law.

Since the UV-Vis range spans the range of human visual acuity of approximately 400 - 750 nm, UV-Vis spectroscopy is useful to characterize the absorption, transmission, and reflectivity of a variety of technologically important materials, such as pigments, coatings, windows, and filters. This more qualitative application usually requires recording at least a portion of the UV-Vis spectrum for characterization of the optical or electronic properties of materials.

Absorption Spectra

Identification of molecules using absorbance spectra

Measurement at a fixed Wavelength

III. Beer-Lambert Relationship

The ultraviolet spectra of compounds are usually obtained by passing light of a given wavelength (monochromatic light) through a dilute solution of the substance in a non-absorbing solvent.

The intensity of the absorption band is measured by the percent of the incident light that passes through the sample:

% Transmittance = (I / I0) * 100%

Where:

I = intensity of transmitted light

I0 = intensity of incident light

Because light absorption is a function of the concentration of the absorbing molecules, a more precise way of reporting intensity of absorption is by use of the Beer-Lambert Relationship:

Absorbance = -log(I / I0) = ecl

Where:

E = molar absorptivity

C = molar concentration of solute

L = length of sample cell (cm)

The longer the pathlength the more light is absorbed. Also, the higher the concentration of compound in solution the more light is absorbed. Absorbance is proportional to pathlength and the concentration (Beer-Lambert's law)

Thus, A = a • b • c where a = absorptivity (epsilon - molar absorptivity includes pathlength and wavelength), b = pathlength (commonly 1 cm), and c = concentration (molar if molar absorptivity)

If b is 1 cm and c is in g/100ml the absorptivity is given as A1%1 cm at wavelength (lambda).

IV. Instrumentation