Colorimetry is the science that describes colors in numbers, or provides a physical color match using a variety of
Beer’s law states that if a monochromatic light is allowed to fall on a solution, the amount of light absorbed or
transmitted is dependent upon the concentration of the species absorbing the light and the length of the light
path through the sample.
Simply, Beer’s Law states that absorbance and concentration have a linear relationship.
As concentration increases, absorbance increases.
o Why? Because there are more molecules interacting with the light passing through the sample!!!
Absorbance = the molar absorptivity constant x the path length x the concentration of the species
A = εbc
A = Absorbance
o Obtained from the spectrophotometer
ε = the molar absorptivity constant; dependent on the substance
b = the path length
o for our purposes, use b=1cm
o This value is the length of the test tube (the length of the sample that light passes through)
c = concentration of the species of interest
Using the Spectrophotometer
Why is the “blank” a test tube filled with water?
o The purpose of the experiment is to use Beer’s Law to determine the concentration of cobalt in a
solution of water held in a test tube.
o We don’t care about the absorbing properties of the water or the test tube. So, we set the
spectrophotometer to zero with the test tube and water to “subtract out” the absorbance of the water
and the test tube.
o “Zeroing” the instrument ensures the absorbance reading is ONLY for the cobalt, and not for any other
species in solution.
How did you come up with 500nm for the wavelength?
o Looking at the color of the solution is a good clue for which wavelength to use.
o Every species has a wavelength at which it has maximum absorbance (λ max).
o Completing an absorbance spectrum for a species will show the wavelength at which the species has
maximum absorbance. To determine an unknown concentration for that species, the sample should be
read at the maximum wavelength.
o When chemists work with an unknown, an absorbance spectrum is run to determine the unknown’s
maximum wavelength. This process helps “narrow down” possibilities of the unknown’s identity.
Determining Unknown Concentration from a Calibration Curve
To determine the concentration of an unknown, we must first construct a calibration curve.
A calibration curve is a plot of known concentrations against the absorbance.
From this data, we can obtain the absorbance of an unknown sample, and determine where it fits into the plot
we constructed of known samples.
For example, using the graph below as a calibration curve, we could determine an unknown sample’s
concentration if it was between 0 and 8 units.
o Let’s say we put the sample in the instrument and we obtained an absorbance reading of 0.4, we could
determine from the calibration data that the unknown concentration is 1.5 units.
Preparing Samples: Serial Dilutions
The stock solution will be made from hydrated cobalt (II) chloride (CoCl2• 6H2O)
o You must prepare a 50.00mL solution of 0.200M CoCl2• 6H2O; this is the stock solution.
o To prepare the solution, transfer 2.379g of the salt to a 50mL volumetric flask.
o Fill the flask to the mark with DI H2O
Complete the dilutions as follows:
With each dilution, you are pulling 25mL of solution from the previous flask, and then filling the volumetric flask to the
mark with DI H2O. Once you have completed this, the process is repeated. This means that the first four flasks will
wind up with only 25mL in them, and only the fifth flask will have 50mL in it.
Laboratory Question 1, page 239