Experiment r:

Spectrophotometric analysis of Kool-Aid Ò

The characteristics of colored solutions have been of
interest to chemists for a long time. Of particular interest has been the fact
that colored solutions, when irradiated with white light, will selectively
absorb incident light of same wavelength but not of others. We can determine the
particular wavelength that the substance will absorb. If light of a particular
wavelength is not absorbed, the intensity of the directed beam at the solution
(I_{o}) will match the intensity of the of the beam transmitted by the
solution (I_{t}). If some of the light is absorbed, the intensity of the
beam transmitted by the solution will be less than that of the incoming beam.
The ratio of I_{t} and I_{o} can be used to indicate the percent
of incoming light that is absorbed by the solution:

_{
}

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 pathlength (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 pathlength is measured in cm, the sensitivity factor is known as the molar absorbtivity (e) of the particular absorbing species. Molar absorbtivity is a proportionality constant of a particular absorbing species with units of L/mol cm. 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. Recall the 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. 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) will give you the mathematical relationship between the two variables. With it, you can calculate the concentration from solution absorbancy.

** **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 be given a sample of Kool-aid which you must mix up with a known amount of water. Measure the absorbance. From your Beer's law graph, you can obtain the concentration of red #40 in the dilution. Knowing how much solution there was (known amount of water) you can determine the number of grams of red #40 in the sample (How do you convert volume to mass of red #40?), and thus mass percent. At least three runs of kool-aid must be done.

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 abs and get equation of resultant line.

**Phase 2:**

1. Take a known mass of solid kool-aid sample and dissolve it into a known
volume of water.

2. Dilute it until you achieve a measurable absorbance.

3. From absorbance found in step 2 and equation found in phase 1, step 3 above,
determine the concentration of red #40 in the diluted sample.

4. Knowing the volume used in step 2, calculate the concentration of the
solution made in step 1.

5. Knowing the volume of water used in step 1, calculate the mass of red #40 in
the step 1 solution.

6. Determine mass% of red#40 in kool-aid sample (mass red #40 found in step 5
divided by mass of kool-aid sample in step 1.

**Lab Write Up**: All of your data (including dilution
information) must be given (in nice tabular form) A brief overview of your
purpose, procedure, results (including sample calculations) and a 'critique' of the learning experience are also
required.
*Also include a one sentence 'suggestion' to a future student who is about to
START this experiment. *
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 mass % of red#40 in the powder as well
as the molar absorbtivity 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

V Stock (mL) |
concentration of dilute solution |
Absorbance |

6.3 |
? |
0.181 |

12.5 |
? |
0.232 |

18.8 |
? |
0.383 |

25.4 |
? |
0.604 |

35.1 |
? |
0.752 |

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

3. The student then took 0.732 grams of a sample of Kool-aid and diluted it in 100 mL of solution. They measured the absorbance to be 0.431. What is the mass % of color in the original (dry) Kool-aid sample?

4. Come up with a data sheet to be shown to your instructor upon entering the lab. No data sheet, no lab.

~MEO 01.26.05 07:57