7.

SPECTROPHOTOMETRY – SPECTROPHOTOMETRY &

BEER’S LAW
JHU INTRO. CHEM. LAB II
SPRING 2019
Alongside emission spectroscopy, spectrophotometers can also be used to measure optical absorption
spectra from various chemical species. The specific wavelengths that are absorbed can be used to identify
species just as you did for the previous metal atomic species. In addition, it is often used to determine their
concentrations in solutions. This is a standard analytical technique that you will likely use again in other
undergraduate labs at Hopkins. In particular, the biochemistry lab uses this technique in many of its
experiments. It is also a commonly encountered research tool. The device you will use to make these
measurements is a spectrophotometer used in absorption mode.
In this experiment, you will use Beer’s law to relate light absorption to concentration. You will use the
SpectroVis Plus spectrophotometer, controlled by the LabQuest2 interface. You will work in your regular
group of 2 students and have one lab period to complete this experiment. Your postlab Chem21labs assignment
includes uploaded graphs.
Objectives
• Make serial dilutions to obtain desired concentrations of solutions. You will need to do this for the
lab skills exercise later this semester.
• Use a spectrophotometer to measure absorption spectra.
o This will include making a calibration curve to determine the relationship between
absorbance and a known concentration using Beer’s law.
o You will use the calibration curve to determine concentrations from measured absorbances.
• Recognize some of the similarities and differences between absorption and emission spectroscopy.

Safety First!
Always wear goggles, gloves, and apron during lab.
The Froot Loops are not fit for human consumption.

BEER’S LAW
There are many ways to determine concentrations of a substance in solution. So far, the only experiences
you may have are acid-base titrations or determining the pH of a solution to find the concentration of hydrogen
ions. There are other properties of a solution that change with concentration such as density, conductivity and
color. Beer’s law relates color intensity and concentration. Using color can be much faster than doing a
titration, especially when you have many samples containing different concentrations of the same substance,
but the tradeoff is the time required to make a calibration curve.
Light passing through an absorbing solution is attenuated according to the relation:
𝐼
= 10−𝜀𝑐𝑙 (1)
𝐼0
l

I0 I

Absorbing Species
(c and ε)

where I is the signal intensity of the attenuated light, I0 is the intensity of the incident light before hitting the
sample, c is the concentration of the absorbing species, and l is the pathlength of the light beam through the
solution (i.e. the cuvette thickness). The molar extinction coefficient, , is a property of the species that
accounts for how much light is absorbed by the species. A sketch of this is shown here:
If the wavelength of the light is varied without changing the concentration or the pathlength, the intensity of
the attenuated light will normally change. Since the concentration and pathlength are constant, the extinction
coefficient is the term that is dependent on the wavelength. Also note the exponential form of this relation,
which is one of its most important characteristics.
Beer’s law states that the optical absorbance, A, of a species in solution is given by:
𝐴 = 𝜀𝑐𝑙 (2)

For fixed wavelength and pathlength, the absorbance is linearly related to the concentration.

USE OF THE SPECTROPHOTOMETERS

The spectrophotometer works by using a diffraction grating to spectrally separate a beam of light which
is then collected and sorted by a charge-coupled device (CCD) array detector. If the sample absorbs light in
the wavelength range, the intensity transmitted at the absorption wavelength will be lower than if no sample
is present. However, it is not only the absorbing species that absorbs light. Both the container holding the
solution and the solvent may also absorb small amounts of light. In some experiments there are also other
light-absorbing species in the solution. It is necessary to determine the total absorbance due to the container
and these other absorbing species and subtract this from the absorbance measured for the sample to determine
the absorbance due to the species of interest. Some instruments divide the light into two beams so both the
reference and the sample can be measured simultaneously. Some, like the instrument you will use in the lab,
just have a single beam, so the reference cell with the other absorbing species and the sample cell must be
measured sequentially. The reference cell is called the “blank”.
When a species absorbs light, the color you see is the color of the light NOT absorbed. The observed color
of a solution is the complementary color of the light absorbed. A color wheel, showing complementary colors
looks as follows:
800 400
nm nm
RED VIOLET
620 nm 430 nm

ORANGE BLUE
580 nm 490 nm
YELLOW GREEN
560 nm
So, for example, if a solution looks yellow, that means that it absorbs in the violet. If a solution looks
green, it absorbs in the red.
The spectra for the species in this experiment, alongside their chemical structures, are shown below:
1.2
Red#40: Allura Red AC
Red#40
1 Blue#1
Absorbance /arb. units

0.8

0.6

Blue#1: Brilliant Blue FCF

0.4

0.2

0
400 450 500 550 600 650 700 750 800
Wavelength /nm
 The spectrophotometer reads absorbances to the thousandths place (0.xxx). That is the accuracy you
should report in your notebook and assignment. The absolute accuracy of the instrument is only to the
hundredths place because there is some drift in the intensity of the light and uncertainty in the wavelength.
However, by making your own calibration curve for the wavelength you choose for your experiment, you
correct for any wavelength uncertainty. Also, by directly checking the value of the absorbance in the blank
during each series of measurements, you make sure that there is little drift in the intensity of the light, thereby
improving the accuracy of your results. Because of this, you should report and use values to the thousandths
place.
In these experiments the subtraction of the absorbance due to the other light-absorbing species is done
electronically by the spectrophotometer by measuring a reference “blank” cell in a calibration run. The
reference cell is made of the same material as the sample cell and has the same wall thickness and pathlength.
It contains the solvent and any other species that absorb light other than the species you are trying to measure.
With the SpectroVis spectrophotometers, this reference “blank” cell is included as the calibration cuvette.
The wavelength chosen for the measurements depends on the species. Ideally it will be near the maximum
of the species of interest and the minimum for any absorbing species in the blank. The plot shown here contains
curves for all potential dyes shown above. Since the spectrophotometer subtracts the absorbance due to the
solvents and the cuvette in the blank electronically, all you will see will be the subtracted spectrum, which
will look like the solid curves. You will be able to determine the best wavelength to use from the maximum
of your spectrum.
For accurate measurements, it is necessary to have the same path length for the blank and all the samples.
In this experiment, you will use special containers known as cuvettes, which are carefully manufactured to
have a standard path length of 1.000 cm. Treat them carefully. Do not scratch them. Use the Kimwipes (not
regular paper towels) to wipe off the outside of the cuvettes if they are wet or dirty.
In this experiment, you will first prepare a set of stock solutions containing your chosen food coloring at
known concentrations. You will plot the absorbance as a function of concentration and determine the
extinction coefficient at the maximum wavelength of your scan. You will then use this plot to determine the
concentration of the dye contained within some Froot Loops®.

PROCEDURE

PART A
• From the stockroom, check out the following equipment:
o 2× 50 ml volumetric flasks;
o 2×100 mL volumetric flasks;
o 3 pipettes (1 ml, 5 ml, and 10 ml) with corresponding pipettors;
o mortar and pestle;
o 500 ml filter flask w/ rubber adaptor and Buchner funnel.
• Cuvettes and caps can be obtained from your TA, you will need 5 of each.
o The cuvettes you will use will be placed between the white light symbol and the arrow
on the spectrophotometer. Handle them carefully using a Kimtech wipe to keep the
clear sides clean and free of scratches/fingerprints.
Caution: If you get any solution on your skin, rinse immediately with water.
The precise concentrations will depend on the actual concentration of the stock solution and how you do the
dilutions. Specific directions will not be given in the manual or by your TA. You are expected to be able to
figure them out on your own. Learning how to do simple dilutions will be important for the lab skills exercise
later in the semester.
• Decide on which color of Froot Loops® you would like to study, red or blue. Record the color.
• Given your choice of loops, obtain the corresponding dye (Red #40 or Blue #1) stock solution.
• Record the name, concentration and color of the stock solution you selected in your lab notebook.
• Rinse out a cuvette with a small amount of solution first and then fill it about ¾-full of the stock
solution.
• Fill another cuvette with a similar amount of distilled water. This will be used as the blank to calibrate
the spectrometer.
 • Cap the cuvette when it is filled and mark the cap so you know which cuvette is which. Dry the
outside of the cuvettes carefully using a Kimwipe.
• Plug the SpectroVis into the USB port on the left side of the LabQuest2. The default reading is in
Absorbance (Abs) in Full Spectrum mode.
• Before beginning your measurements, the spectrophotometer needs to be calibrated with the blank,
using the cuvette you have prepared for this. To do this, tap the red box, tap “calibrate” on the pop-
up menu, and follow these instructions:
o Wait for the spectrophotometer to warm up; only complete this step for the initial
calibration, it can be skipped if re-calibration is necessary later-on.
o Once warm up is complete, place the blank inside the spectrometer and select “Finish
Calibration”.
o Press the “OK” button once calibration is complete.
Once calibrated you need to collect spectra at a series of concentrations between 0 and the concentration of
the stock solution. Record the spectrum of the stock solution first, this will be solution 1.
• Place the cuvette with the stock dye in the spectrophotometer and click on the green arrow to start
the data collection.
• To record a spectrum, click “stop” when you are happy with how the data looks.
• Draw a rough sketch of this spectrum in your lab notebook and note the maximum wavelength (λmax).

PART B
• Determine (with calculations) how to make at least four dilutions of the original solution using 5-,
10-, 20- and 25-ml pipets and your two 100-ml and two 50-ml volumetric flasks that will give well
spread out data points on the calibration curve.
• Make the required solutions as accurately as possible.
• Collect absorbance data for the diluted solutions as you did for solution 1. It is worth noting the
following:
o Be sure to record each of the spectra as a new run (hit the filing cabinet icon) as you
will need to submit a graph showing all of your data.
o You should write down the absorbance reading at your chosen maximum wavelength
of your cuvettes, including the blank, just in case something goes wrong with your
spectra.
o The blank should read 0, but as long as it reads 0 ± 0.005 it is fine. If the blank is outside
this range, you should recalibrate using the blank and repeat all the trials.
• At this point, sketch a plot of absorbance (at your chosen λmax) as a function of dye concentration in
your notebook. This should be linear with points ranging from 0 to ~1.2 dependent on the stock and
dilutions you made. If it is not, you should repeat data points, making new mixtures if necessary.

PART C
• Collect approximately 10 Froot Loops® rings that match the food dye that you used in Part A and B
of the experiment.
• Accurately (analytical balance) determine the mass of your rings and record it in your notebook.
• Grind the rings to a fine powder with a mortar and pestle.
• Transfer powder to a 150-mL beaker. Rinse the mortar and pestle into the beaker with a little water
from your wash bottle.
• Measure 25.0 ml of distilled water in a graduated cylinder and add it to the Froot Loops® powder
into a 100 ml beaker.
• Using a hot plate, heat, with stirring, the Froot Loops® slurry until it just starts boiling.
• Turn off the hotplate, remove the beaker, and let it cool to room temperature.
• Add 25.0 ml of acetone to the cooled slurry.
• Stir the slurry/acetone mixture on a cooled stirrer-hotplate until the solids settle easily and give a
clear (not colorless) solution. This should be done for at least 5 minutes.
 There is likely to be a small amount of cloudiness to your solution, if you suspect it is too cloudy, ask your
TA for advice.
• After letting the mixture settle, decant and filter the solution in the beaker using the Buchner funnel.
Keep as much of the solids in the beaker as possible to prevent the filter from clogging. Remember,
that you want to collect the filtrate so ensure that the Buchner flask is clean.
• Rinse the remaining solids in the beaker once with a few ml of 1:1 acetone/water.
• You will need to dilute this extracted solution up to 100ml using a volumetric flask in order to collect
an accurate value for the absorption. Make sure to keep track of the volume of 1:1 solution added
such that you can accurately calculate the concentration.
• Measure the absorbance of the extracted solution as you have done previously. You should re-check
the blank and recalibrate if necessary.
• Once again, be sure to record the absorbance in your lab notebook.
• Make sure to save all of your data and either email it to yourselves or transfer it to the USB drive
from your TA desk.

EXCESS CHEMICAL DISPOSAL

Stock dye solutions can all be disposed of in the large white drum. Dyes extracted from the cereal contain
acetone; therefore, they should be disposed of in the red can labeled “Organic Waste” located in the large
fume hoods.

DATA ANALYSIS
For your data analysis you are attempting to calculate the approximate concentration of your chosen
dye per gram. In order to do this, you will use Beer’s law alongside the calibration curve you generated in Part
B.
First, you must make a calibration curve by plotting Abs versus concentration. Include the four
dilutions, the blank (an absorbance of ~0.0 at 0.0 M concentration) and the original solution, for a total of 6
data points. Determine the molar absorptivity, ε, from the slope. Then, calculate the concentration of the
solution in Part C using Beer’s Law. Finally, knowing the concentration and volume of the solution, the molar
mass of the dye, and the mass of the Froot Loops sample, you should be able to calculate the mass percent of
the dye in your sample.