Spectrophotometric analysis of Colored soda.

The wavelength at which the percent transmittance (%T) is lowest is the wavelength to which the solution is most sensitive. This wavelength, which is the one we will use for analysis, is called the analytical wavelength.

            Once we determined the analytical wavelength for a particular solution, we can study the three variables that influence the specific response of the solution. These variables are the concentration (c) of the absorbing substance in the solution, the path length (b) of the light through the solution, and the sensitivity of the absorbing species to the energy of the analytical wavelength. When concentration is expressed in molarity (mol/L)and the path length is measured in cm, the sensitivity factor is known as the molar absorptivity (e) of the particular absorbing species. Molar absorptivity is a proportionality constant of a particular absorbing species whose units depend on the units of concentration. Its value depends of the analytical wavelength used for the analysis. The product of these three variables is absorbance (A) :

A = ebc

A relationship known as Beer's Law. Thus we cab define absorbance in terms of I_o and I_t:

A spectrophotometer is an instrument used to study the response of solutions to light. There are two different way of measuring how much light interacts with the species in questions, % transmittance and absorbance.

A = 2.000 – log (%T)

    We can see from the Beer's law equation, the absorbance is directly proportional to the concentration of the absorbing substance in solution. If we use containers of consistent size (called cuvettes) we can measure the absorbance of a series of (known) concentrations of solutions and create what is known as a Beer's law plot. The linear relationship between concentration and absorbance will allow us to get an equation of this relationship (the best fit line from our Beer's law data) Spectrometers work best (and Beer's law is linear) when the data is in the range of 10-90 % transmittance. (Absorbance range of 0.05 to 1.0)

            In this experiment, you will be given a stock solution of a particular FD&C color. This solution is much more concentrated than will work in the spectrometer, so you will have to dilute it very carefully. The following is known as the 'dilution equation':

CsVs = CdVd

Where Cs is the concentration of the stock solution, Vs is the volume of the stock solution and Cd and Vd are concentration and volume of the diluted solution respectively. The concentration of the stock (Cd) will be given to you. You will measure the amount of stock solution you put into a volumetric flask. You will then dilute it to a known (diluted, so Vd) volume. That leaves only the calculation of Cd.

All dilutions will be done with a graduated pipets and volumes should be measured to 2 decimal places.

You will make solutions of varying concentrations, getting absorbencies over the effective ranges (0.05 and 1.0) of the spectrometer. A pretty Beer's law graph (concentration vs. absorbance, made using this technique) will give you the mathematical relationship between the two variables. With it, you can calculate the concentration from solution absorbency.

 You need to make a series of solutions that are varying in concentration and give absorbencies in the range of 0.05 and 1.0 and points in between. How many should you make? It is your choice, but the more, the better. Once you have data that you think is good for your Beer's law plot (maybe graph it right then and there?)

You will then test your unknown solution (A red colored soda) which you will test the absorbency of. If the absorbency is too high, you will have to dilute it (that's ok, you can back-calculate the concentration of the original solution easily enough) From the measured value of absorbency, you can calculate the concentration of color in your soda sample.

A different way of thinking about the experiment is to break it up into 2 phases:

Phase 1:
1. From a a known concentration stock solution make several different ones
2. Measure absorbencies of solution in step 1
3. Plot concentration versus absorbance and get equation of resultant line.

Phase 2:
1. Measure the absorbance of your soda sample solution.
2. If too concentrated, dilute it until you achieve a measurable absorbance.
3. From absorbance found for the soda sample and equation found in phase 1, step 3 above, determine the concentration of the color in the unknown sample.
4. Knowing the volumes used in step 2, calculate the concentration of the original (undiluted) soda sample made in step 2 (if needed)
 
8 minute screencast of the experiment

Lab 'Picture':

 Lab Write Up: Short form memo. A nice graph of your Beers' Law data must also be included. Here is a nice little tutorial on making nice graphs that include the equation of the best fit line. You must report both the concentration of red#40 in the soda as well as the molar absorptivity of red #40.

 Prelab Questions: (to be completed on a labeled piece of recycled paper)

1. What is the formula for FD&C red #40 (also known as Allura Red AC)?

2. A student did this experiment, given a stock solution that was way too concentrated, 1.453 mg Red#40/L. The stock solution was then diluted according to the following schedule, (all diluted to 50 mL) and absorbencies taken of the new solutions. Calculate the concentrations of the individual solutions using the dilution equation above.

Prepare a computer generated plot of the absorbencies of each of the solution (y axis) vs. concentration in mg/L (x axis). Print this off on a recycled piece of paper (if possible)

3. The student then took a unknown soda solution and measured the absorbance to be 0.431. What is the concentration (in mg/L) of color in the soda?

4. Come up with a data sheet (on a separate piece of recycled paper) to be shown to your instructor upon entering the lab. No data sheet, no lab.

~MEO  9.25.07