Visible Spectra of FD&C dyes and Beer’s Law
Visible Spectra of FD&C dyes and Beer’s Law
Objective: To identify a substance by its absorbance spectrum and to discover the
relationship between absorbance and concentration.
I give these instructions verbally to the students, no written instructions are
handed out.
Each team in in charge of preparing a working solution of one dye (of known
concentration in units of grams per Liter.)
FD&C Dyes: Green 3, Red, 3, Red 40, Yellow 6, Blue 1, Yellow 5
Part I- Visible spectra of Dyes
Make sure you write the procedure in your notebook as you do the
experiment, you will need it for your lab report write-up.
1. Make a solution of your dye by accurately weighing between 0.010 to 0.030
grams of dye and dissolving in a 100 mL volumetric flask, record amount in
your lab notebook. Mix vigorously. This is your stock solution.
2. Calibrate the spectrometer using deionized water. (Give explanation of a
spectrometer and how it works)
3. If the absorbance of the dye is above 2.0, dilute by taking an aliquot of the
dye and dissolving it in another volumetric flask (either 100 or 50 mL) with
deionized water. Record in your notebook the dilution, calculate the
concentration and label the solution accordingly.
Make sure you label all your solutions.
Determine the visible spectrum of a dye using the Vernier spectrometers:
1. Calibrate your spectrometer with deionized water if not done yet.
2. Set the spectrometer to give you an absorbance vs. wavelength spectrum
3. Find the absorbance maxima of the dye (max), the point with highest
absorbance (peak) and record in your notebook.
Design a table to record the absorbance at max of all the dyes.
Once you have prepared the table, borrow a small sample of each team’s diluted dye
and observe the spectrum of that dye, record the Absorbance at max for that dye in
your table.
Take your unknown and measure the absorbance spectrum. Based on the max in
your table and the spectrum of the unknown, can you determine which dyes were
used to make the unknown?
Transfer your stock dye solution to an Erlenmeyer flask and cork. Label your dye
solution with the dye color and number, your names and the date and store for next
period.
Part II
Absorbance vs. concentration
Use your stock solution to make a dilute solution of your dye (absorbance at max
less than 2.0) based on your part I results.
Make a series of solutions from your working solution of dye by measuring for
example 1, 3, 5, 7, and 9 mL of stock into 10 mL volumetric flasks and diluting with
deionized water to the mark. Mix well. (Each team is given two 50 mL volumetric
flasks and three 10-mL volumetric flasks)
Determine which dilutions you will be using for each one of your solutions and
determine the concentrations in g/L
Set up a table with a column containing the dilution and concentration of each
solution in separate columns. Add a column for the absorbance at max
Choose the max for your dye, measure and record the absorbance for your samples
in your table.
Using Excel plot absorbance vs. concentration in grams/Liter. Find the equation of
the line, the intercept should be very close to 0.0. The slope of the line is
characteristic to the dye, we will talk about it in class.
Determine the concentration of the unknown.
Hand out given to students:
Write a full lab report for this lab.
For the title discuss with your team an appropriate title for this lab report and each
team member should use the chosen title.
For the introduction make sure you include the following:
1. Give information about the dyes used.
2. Describe a visible spectrometer and how it works.
3. Make sure you cite all your sources.
For the experimental section include the following:
1. List the chemicals used (dyes)
2. List the glassware used
3. Describe the experiment as you carried it out so it can be replicated.
For the results and discussion
1. Use a narrative to describe and discuss what you found out and refer to
graphs and tables by number. Make sure you refer to all the figures and
tables included in your discussion.
2. Use excel to make the tables and graphs. Label graphs as figures for example
“Figure 1. Absorbance spectrum of dye No.4“ at the bottom of the graph.
Tables should have a title at the top of the table for example “Table 1.
Absorbance maximum of different dyes”. All tables and graphs should be
integrated in the document.
3. Show all your calculations
For the Conclusion
Summarize what you found and talk about what you learned.