Lab Experiment #361
Spectrophotometric Analysis of Food Dye Solutions

 

We can determine the particular wavelength or group of wavelengths absorbed by systematically exposing the solution to monochromatic light of different wavelengths and recording the responses.

 

Io = intensity of beam directed at solution.
It = intensity of beam transmitted by solution.

 

  • If a particular wavelength is not absorbed, Io will match It.
  • If some of the light is absorbed, It<Io

Therefore, the ratio of It and Io can be used to indicate the percent of incoming light that is absorbed by the solution. This is calculated by percent transmittance (%T)

%T= It/Io x 100

 

The wavelength at which percent transmittance (%T) is lowest is the wavelength to which the solution is the most sensitive. This wavelength is called the analytical wavelength.

 

Once the analytical wavelength is determined for a particular solution, we look at the three variables that influence the specific response of the solution:

 

1. Concentration (c) of the absorbing substance in the solution
2. Pathlength (b) of the light through the solution

3. Sensitivity of absorbing species to the energy of the analytical wavelength

 

 

Molar absorptivity (E) is sensitivity factor expressed in concentration molarity (mol L-)  and the pathlength is measured in cm.

 

 

Absorbance (A) is dependent on the three factors Ebc, thus: A = Ebc

Absorbance is equal to (molar absorptivity)(concention)(pathlength)

This relationship is known as Beer's Law, and we define absorbance in terms of Io and It as:

A = log(Io/It)

 

Using a spectrophotometer, we must convert readings on percent transmittance scale to equivalent absorbance using the equation A =2.00-log(%T)

 

Concentration is determined using the following equation:

                                 concentration, drops mL^-1 = # drops/volume of solution in mL

Meaning that for every  X drops of dye, Y mL of solution were added, for example, if 1 drop of dye was added to 40mL of solution:

concentration, drops mL^-1 = 1 drop/40mL = 0.025 or 2.5 x 10^-2 drops per mL^-1

 

More dilute solutions were made using solution #1 and distilled water, the following equation is used to calculate their concentrations:

 

concentration of diluted solution, drops mL^-1 = (vol solution 1)(concentration solution 1)/total volume diluted solution

 

Which is to say that we take the volume of solution one that we are planning to mix to make solution two and multiply it by the concentration of solution one, then divide the result by the volume of solution one that was mixed with the volume of solution two. That sounds aaaaaaaaaawfully confusing, even to me, and I wrote it!

It best understood when given in example format.

 

So we have solution one, which was 1 drop in 40 mL which equates to 2.5 x 10^-2 mL^-1, that is the concentration of solution one.

To prepare solution two, let's say we take 5.00mL of solution 1 and dilute it with 5.00mL of distilled water. To afterwards calculate the concentration of solution 2, we would multiply the volume of solution one taken to make solution two by the concentration of solution one:

(5.00mL)(2.5x10^-2 drops mL^-1) = 12.5 x 10^-2 drops mL^-1

Then divide the answer by the total volume, so 5.00mL solution one plus 5.00mL distilled water = 10.0mL total, therefore:

12.5 10^-2 drops mL^-1/10 =1.25 x 10^-2 drops mL^-1

So our concentration for solution two is 1.25 x 10^-2 drops mL^-1

 

We can continue to use this equation for all subsequent solutions, using the concentration of the previous solution to calculate the concentration of the next solution.