Spectrophotometry Theory

Effect of Wavelength of Light
The nature of the interaction between light and matter depends on the wavelength of the light and the structure of the matter. Useful wavelengths for spectrophotometry range from 185 to 3,000 nm. Absorption of longer, low energy wavelengths (infrared) stretches and bends bonds in molecules. The resulting complex patterns of absorption at different wavelengths are used in IR spectroscopy to identify chemical structures. Shorter wavelengths (X-rays and gamma rays) are sufficiently energetic to strip electrons from molecules. In the visible/UV range used in spectrophotometry, molecules absorb light energy with the movement of electrons to a higher orbital. Note that molecules absorb only those wavelengths of light that contain just the right amount of energy to move them to a higher energy state, resulting in peaks and valleys when absorbance is plotted against wavelength.

Effect of Concentration of Substance
The amount of light passing through the sample decreases exponentially (by percentages) as the concentration increases.

Figure One

This relationship will be clear if you think of two equivalent cuvettes in a series. (Mouse over the Figures to see a full picture)

Figure Two

The first cuvette absorbs 90% of the incident light. The second cuvette absorbs 90% of the light passing through the first cuvette, so that only 1% of the light incident on the first cuvette reaches the photocell. Using two cuvettes has the same effect as doubling the concentration of the light-absorbing substance in the first cuvette.

Spectrophotometers present the results either as percent transmittance or as absorbance. Percent transmittance is simply the percentage of incident light reaching the photocell:

%T = 100 * (I / Io) where I is the light reaching the photocell and Io is the light striking the cuvette.

The exponential decrease of transmittance with concentration is awkward to work with: straight lines are more convenient than curved ones. Since transmittance decreases exponentially with concentration, log 1/T increases linearly with increasing concentration (See Figure 3).

Figure Three

The second scale on the spectrophotometer reads absorbance (A), according to the equation A = log 1/T, with T expressed as a decimal fraction. This can also be expressed as A = - log T or A = 2 - log %T, with T expressed as a percentage. The relationship between absorbance and concentration is known as the Beer-Lambert law: A = elC , where e is the extinction coefficient (a property of the light-absorbing substance), l is the light path in cm and C is the concentration of the light-absorbing substance. Most cuvettes have a light path of 1 cm.

On to Operating Guide