Spectroscopic Measurements and the Beer-Lambert Law

When a beam of radiation (light) passes through a substance or a solution, some of the
light may be absorbed and the remainder transmitted through the sample. The ratio of the
intensity of the light entering the sample (Io) to that exiting the sample (It) at a particular
wavelength is defined as the transmittance (T). This is often expressed as the percent
transmittance (%T), which is simply the transmittance multiplied by 100. The absorbance
(A) of a sample is the negative logarithm of the transmittance.

% T = (Io / It ) x 100

A = - log (T)

The absorbance of a sample at a given wavelength is proportional to the absorptivity of

the substance (a constant at each wavelength), the path length (the distance the light
travels through the sample) and, in many instances, the concentration of the absorbing
substance. In these cases the Beer-Lambert Law holds:

A = a * b * c where a = the absorptivity of the substance

b = path length
c = concentration of the substance

Commonly, a and b are constant for a given set of experiments so that a plot of the
sample absorbance vs. the concentration of the absorbing substance is a straight line. In
practice, a calibration curve is prepared by plotting the absorbance of a series of standard
samples as a function of their concentration. If the absorbance of an unknown sample is
then measured, the concentration of the absorbing component can be determined from
this graph.

UV-Visible spectrometers

UV-Visible spectrometers are composed of four basic parts: the light source, a
monochromator, the sample holder and detector. The most commonly used light sources
produce a continuous spectrum of radiation. As the Beer-Lambert Law does not hold for
multiple wavelengths, a monochromator (historically this contained a prism, but currently
gratings are used) is used to select a single wavelength of light. The light leaving the
monochromator is directed through the sample cell which contains the solution to be
analyzed. A detector (normally a photo- or photomultiplier tube) is used to determine the
intensity of the radiation passing through the sample.
 A schematic of a visible light spectrometer.
The wavelength of light is selected by rotating the grating.

A series of standard solutions containing a red dye was made by diluting a stock solution
and then measuring the percent transmittance of each solution at 505 nm (greenish blue).
This wavelength was selected by examining its absorption spectrum. If the solution looks
red, it is absorbing red's complementary color of light, which is greenish blue. The
results, after conversion to absorbance, are shown below.

An absorbance of 0.39 was also determined at 505 nm for a solution with an unknown
concentration of the red dye.

solution concentration absorbance

blank 0.00 M 0.00
standard #1 0.15 M 0.24
standard #2 0.30 M 0.50
standard #3 0.45 M 0.72
standard #4 0.60 M 0.99
sample ???? M 0.39

If the absorbance of the blank and standards are plotted as a function of their
concentration we call it a calibration or standard curve. Note that the points form a
straight line.
The equation for the best fit line through the points is

y = 1.64 x - 0.002

Substituting the meaning of x and y into the equation tells us that:

absorbance = 1.64 * concentration - 0.002

To solve for the concentration of the dye in the unknown simply plug in the absorbance
value:

0.39 = 1.64 * concentration - 0.002

and solve for the concentration. In this case it is equal to 0.24 M.