Experiment 37B-2

Draft 3/23/14

SPECTROSCOPIC ANALYSIS OF DYES – MORE THAN PRETTY COLORS1

MATERIALS: FD&C food stock solution (Red), 100 mL beaker, volumetric pipets (one each of 1, 2, 5, 10
mL), five 100 mL volumetric flasks, Spectronic 20, 6 cuvettes, unknown.

PURPOSE: The purpose of this experiment is to understand how light interacts with a dye solution and
to use this knowledge to determine the amount of dye in an unknown.

LEARNING OBJECTIVES: By the end of this experiment, the student should be able to demonstrate the
following proficiencies:

1. Prepare diluted solutions and calculate their concentrations.

2. Understand what an absorbance spectrum is and select max.
3. Relate the color observed of a solution to its max value.
4. Understand Beer’s Law and what factors affect it.
5. Create a calibration curve using standard solutions.
6. Determine a concentration of an unknown using a calibration curve.

PRE-LAB: Complete the pre-lab questions on page E37B2-5 before lab.

DISCUSSION: Mars

Colorimetric analysis, or colorimetry, is an important analytical technique in chemistry. It allows an analyst

to quantify the amount of a substance of interest (analyte) in a solution based on color properties. Colorimetric tests
are not just used in the chemistry lab. For instance, it is important to quantify the amount of chlorine present in a
swimming pool. Pools are chlorinated to disinfect the water. If chlorine levels are too low, harmful microorganisms
can thrive. If the level of chlorine is too high, the water can be irritating. Chlorine test strips are commercially
available. By comparing the test strip to a set of known standards, it is possible to quantify the chlorine in the pool
and adjust the pool chemistry as needed.

While sometimes it is sufficient to determine only whether an analyte is present or the approximate amount
of analyte present, it is important to be able to measure a precise amount of analyte present in a sample. The
quantitative relationship between absorbance and concentration is one method to determine the amount of analyte
present. This relationship takes the form of Beer’s law. Beer’s law states that

A = lc

where A is the absorbance of the solution,  is the molar absorptivity of the analyte, l is the path length of the
spectrophotometric cell, and c is the concentration of the analyte in the solution. By constructing a Beer’s law plot of
absorbance vs. concentration, the value of l can be determined; it is the slope of the line. Because the path length of
the spectrophotometric cell can be measured, it is possible to calculate the molar absorptivity, , of the solution.

The molar absorptivity () is an important proportionality constant. If you measure the absorbance of a solution of
unknown concentration, then you can determine its concentration by rearranging Beer’s Law and solving for
concentration. To do this, the value of molar absorptivity must be known. Molar absorptivity depends on
wavelength, so it is important to conduct all of your analyses at the same wavelength. Beer’s law plots are frequently
used in colorimetric analysis because they provide the simple relationship between absorbance and concentration.

To compare two solutions, it is not necessary to measure the entire absorbance spectrum. Rather, absorbance (or %T)
can be measured at a specific wavelength. Usually, max is chosen because it produces the maximum response from
the spectrophotometer. In other words, it is the wavelength at which absorbance is the most sensitive to
concentration changes. This allows you to work with less concentrated solutions.

1
This lab is based on “Spectroscopic Analysis of Food Dyes” by Barbara A. Reisner, Joycette Santos-Santori, Dawn
Rickey, and Melonie Teichert.
E37B2-1
 In this part of the experiment, you will measure the absorbance of four “standard solutions” of known concentration
and construct a Beer’s Law plot from your data. Finally, you will determine the amount of FD&C Red No. 3 dye
contained in a sample of a powdered drink mix or a prepared sample.

PROCEDURE:

Work with a partner.

Part A. Preparation of Standard Solutions

You will start with a solution of Red Dye No. 3 with a concentration of 1.36 x 10-4 M. This is the stock solution. You will
use this solution to prepare Standards A – D. (Pre-rinse pipets as necessary.) Both partners should be preparing the
solutions to save time. Part of your lab grade will be based on the accuracy of determining the concentration of Red
Dye in your unknown so work accurately and precisely.

1. In a small dry beaker, obtain about 25 mL of the stock Red dye solution.
2. Use a volumetric pipet to transfer 10.00 mL of the stock solution to a 100.00 mL volumetric flask. Let the pipet tip
drain freely, touching it to the side of the flask. Do not blow out the liquid remaining in the tip. Add distilled water
to the mark, using a dropper to reach the fill line. Stopper the flask and invert it at least 5 times to mix the solution.
Label this Solution A.

Calculate the concentration of Solution A (in units of molarity, report to 3 significant figures):

3. Use a pipet to transfer 7.00 mL of the stock solution to a 100.00 mL volumetric flask (use the 5.00 mL and 2.00 mL
pipets). Add distilled water to the mark, using a dropper to reach the fill line. Stopper the flask and invert it at least
5 times. Label this Solution B.

Calculate the concentration of Solution B:

4. Use a pipet to transfer 5.00 mL of the stock solution to a 100.00 mL volumetric flask. Add distilled water to the
mark, using a dropper to reach the fill line. Stopper the flask and invert it at least 5 times. Label this Solution C.

Calculate the concentration of Solution C:

5. Use a pipet to transfer 2.00 mL of the stock solution to a 100.00 mL volumetric flask. Add distilled water to the
mark, using a dropper to reach the fill line. Stopper the flask and invert it at least 5 times. Label this Solution D.

Calculate the concentration of Solution D:

6. Transfer each of your prepared standard solutions and the unknown to 5 separate cuvettes, pre-rinsing each cuvette
(the round cuvettes are used here). Each cuvette only needs to be about 2/3 full with sample. Make sure you know
which solution is contained in each cuvette. Place the cuvettes in a test tube rack.
7. Fill one cuvette with distilled water after pre-rinsing the cuvette.
8. As an estimate, compare the color intensity of your unknown with the standards. Using a white background behind
your solutions should help distinguish intensities. Estimate a concentration for the unknown based on this
comparison. Record your guess on the data table.
9. Make sure to wipe off the outside of each cuvette with a soft tissue to remove fingerprints or liquid.
E37B2-2
Part B. Absorbance Measurements
1. Set the Spectronic 20 to the max value for the Red dye shown in the prelab by turning the Wavelength knob. Record
the wavelength used. Do not change the wavelength after this is set.
2. Set the Spect20 to the %Transmittance Mode by pressing the Mode button.
3. With nothing in the sample compartment and the lid closed, set 0% T with the left knob (labeled 0% T).
4. Place the distilled water cuvette (the blank) into the sample compartment, close the lid and set 100% T with the right
knob (labeled 100%T).
5. After removing the blank and with the lid closed, the signal should go back to 0%T. The Spect20 has been
calibrated. Spect20’s require recalibration with a blank whenever the wavelength has changed or the knobs have
been moved.
6. Set the Spect20 to the Absorbance Mode. With no sample, the display will be flashing. In Absorbance Mode, the
sample absorbance reading will be displayed directly on the monitor. There are no units for absorbance.
7. Measure and record the absorbance of each of your solutions.
8. Make sure your data make sense before discarding your solutions. Should absorbance increase or decrease with
increasing concentration? Does the absorbance for your unknown fall within the range of the absorbances of the
standards? Does the value make sense?
9. Clean up all glassware and return them to their proper locations. Dye solutions can be disposed down the drain with
water. Make sure to rinse out the cuvettes well. Turn off the Spect20.
10. Start working on the calculations.

E37B2-3
Name ______________________________ Section ___________________________

Lab Partner _____________________________

DATA AND ANALYSIS

Experiment 37B-2

used = __________________________________

Solutions Concentration (M) Absorbance at 

Standard A

Standard B

Standard C

Standard D

Unknown Your Guess 

1. Using Microsoft Excel, construct a Beer’s Law plot of Absorbance (y) vs Concentration (x) using the 4 standards.
Label the axes. This Beer’s Law plot is also known as a Calibration Curve.

2. Construct a trendline through the data. Include the equation of the line and R2 value on the plot.

Equation of the line  _______________________________________________________

If the pathlength (l) of the cuvette is 1.00 cm, what is the value for the molar absorptivity () for this Red dye at
max? Report the value with units.
 = _______________________________________

3. Given the absorbance for the unknown, solve for the concentration of the unknown. Use your equation of the line
above (not the Beer’s Law equation which assumes a zero y-intercept). Show your work below.

4. How close was your guess to the calculated concentration of the unknown?

5. Explain the purpose of a calibration curve in a quantitative analysis.

E37B2-4
 Name ______________________________ Section ___________________________

PRE-LAB QUESTIONS - EXPERIMENT 37B-2

Complete these questions before lab.

1. a. Read the lab procedure and calculate the concentrations of Red dye Standards A-D (which you will prepare in
lab).
Show your work on page E37B2- 2, and record your final values here (to 3 sig figs, and with units).

Concentration of Standard A = _______________________________

Note: these
Concentration of Standard B = _______________________________
concentrations are quite
small. Not much dye is
Concentration of Standard C = _______________________________
needed to see a color.
Concentration of Standard D = _______________________________

b. Inspect the absorbance spectrum for red dye No 3 shown below. What is the approximate value of the max, that
is, the wavelength at which the dye absorbs most strongly? Include units.
max = ________________________

c. How do you predict the absorbances of each of the 4

standards will compare at max for the Red dye? Will they be the same
or different? Explain.

d. How do you predict the absorbances of each of the 4 standards will compare at 650 nm? Will they be
the same or different? Explain.

2. Examine the Beer’s law plot on the right and use the
information provided to obtain the extinction
coefficient, , for salicylic acid. Assume a pathlength
of 1.0 cm. Provide the correct units for .

E37B2-5