RoHS/WEEE HowDoes Spectrophotometry

How does Spectrophotometry work?

Spectrophotometry works by measuring the relative amounts of radiant flux at each wavelength of the spectrum.

Light is a form of energy, and this energy is inversely proportional to wavelength (the longer the wavelength, the lower the frequency or energy of the photon stream.)

Photons of light can interact with electrons in molecular orbitals and cause the energy content of the atoms or molecules to increase. Depending on the energy (or wavelength) of the photons, different processes can occur, including simple absorbance, reflection, scattering, fluorescence & luminescence (absorption of energy followed by emission at a lower energy), and photochemical reaction (absorbance with bond breakage). This explanation will focus on the simple absorbance process.

Electrons in molecular orbitals can interact with photons of light.
The energy of the photon can move an electron from a lower energy level to a higher energy level (i.e., energy is absorbed).

Measuring the absorption of energy
As depicted above, these energy transitions should result in monochromatic (very, very narrow) absorbance bands at energies (or wavelengths) that are characteristic of the difference in energy levels of the analyte. However, these 'line spectra' normally cannot be obtained due to other transitions with different energies that may be occurring in the molecule, plus solvent-solute interactions, and the difficulty of directing truly monochromatic light into the sample. Therefore, what normally results in UV-visible spectrophotometry is a broader band that is an 'average' of all these energies.

Since the UV-visible transitions involve the electronic states, the absorbance is characteristic of the atom, rather than the state of its neighborhood so it cannot be easily used to identify molecules. And with the 'averaging' of different transitions as noted above, as well as the relatively narrow energy range of the electromagnetic spectrum being used, we normally do not observe bands that are considered characteristic of a particular molecule as we do in IR and NMR.

Electromagnetic Spectrum

Due to these phenomena, UV-visible spectrophotometry is normally used for quantitative analysis rather than qualitative analysis. However, that is also the advantage of UV-Vis over IR and NMR for quantitative analysis. UV-vis spectrophotometry is not limited to just organics and organo-metallics, but is widely used to measure the amount of metallic (like chrome, iron, tin, nickel, etc.) and non-metallic (like ammonia, cyanide, etc.) molecules as well. This advantage is enhanced by the fact that UV-Vis spectrophotometry adheres very well to Beer's law ? one of the fundamental laws of photometry.

The Beer-Lambert Law
Light, before it goes through a sample, starts with initial intensity or energy, (I₀), and emerges from the sample with a lesser intensity (I).

The Transmittance of the sample is defined as the ratio of the emerging intensity to the initial intensity. The highest actual transmittance (barring, of course, any light-emitting processes such as fluorescence), is 1.

Transmittance relates to sample concentration in the logarithmic fashion and is not convenient to use for concentration measurements. Absorbance, which is defined as the base 10 log of 1 divided by the transmittance offers the advantage of being linear with concentration.

The equation, commonly known as the Beer-Lambert Law or Beer?s Law provides quantifies this relationship. Beer?s law is typically expressed as:

A = abc, where:
'A' or Absorbance is the absorbance of a sample
'a' is the absorptivity (the physical characteristic of a molecule being analyzed).
'b' is the path length that the light travels through the sample
'c' is the concentration of the analyte in the sample

Light going through a sample

This simple equation is the reason that UV-Vis has been such a widely-used technique, both in working laboratories and in teaching labs. It is easy to understand and requires only simple arithmetic. Indeed, as electronic technology improved, spectrophotometers could output the data as a final result in concentration, thereby eliminating the need for the analyst to perform the calculation.

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