Chemistry 1215 Experiment #11
Spectrophotometric Analysis of an Unknown Brass Sample
In this experiment you will use spectrophotometric measurements to determine
the copper concentration of a solution prepared from a sample of solid brass. Then, using
the concentration of the copper you will determine the percent copper in the brass
Electromagnetic radiation, of which ultraviolet and visible light are but two
examples, has properties of both waves and particles. When light acts as a particle, called
a photon, each light particle possesses a discrete amount of energy called a quantum.
When a molecule is exposed to electromagnetic energy it can absorb a photon, increasing
its energy by an amount equal to the energy of the photon. The energy of the absorbed
photon can be calculated if the frequency, ν, (the symbol, ν, is a Greek letter, pronounced
nu) of the light is known according to Equation 1.
E = hν
Where h is a constant known as Planck’s constant after Max Planck, the German scientist
who first proposed it. Planck’s constant has a value of 6.63 X 10-34 J.s. Frequency is
measured in units of 1/s or Hertz (Hz). The frequency of a light wave is inversely
proportional to its wavelength, λ, (λ is also Greek and is pronounced lambda) which is
typically measured in meters. The product of ν and λ is the speed of light, c as shown in
Equation 2. c has a value of 2.998 X 108 m/s.
c = λν
Molecules are highly selective in the wavelengths of light they can absorb. The
photons absorbed depend on the on molecular structure and can be measured by
instruments called spectrometers. The data obtained from a spectrometer are very
sensitive indicators of molecular structure and concentration.
UV-visible spectrophotometry (UV-VIS) uses only the ultraviolet and visible
regions of the electromagnetic spectrum. UV light ranges from approximately 10 nm to
about 400 nm. The visible region of the spectrum ranges from 400 nm to 700 nm. Red
light lies at the low energy end of the visible spectrum and violet lies at the high energy
end. UV-VIS spectroscopy depends on transitions of electrons in a molecule from one
electronic energy level to another. It is used mostly in studying transition metal
complexes and conjugated π systems in organic molecules. One of the principal uses of
UV-VIS spectrometry is in determining the concentration of an absorbing (colored)
molecule. The amount of light absorbed, and thus the intensity of the color of the
solution, depends on the concentration of the absorbing species in the solution. UV-VIS
absorption peaks are typically quite broad and are often spoken of as bands rather than as
peaks. The wavelength at maximum absorption is referred to as λmax and is the optimal
wavelength for a pure solution. For an impure solution, a “blank” solution can be
prepared which contains all components of the solution except the one being analyzed.
This solution is used to subtract out absorbance due to interfering species.
When a beam of light with intensity, Io, passes through a solution, a colored
species, or analyte, will absorb some of the light energy. The beam of light that passes
through (or is transmitted through) the solution will have a lower intensity, I, than the
incident light, Io because some of the light will be absorbed. Spectrometers typically
measure either transmittance, T, which is the amount of transmitted light, or absorbance,
A, which is a measure of the light absorbed. Both transmittance and absorbance are
measures of the amount of light that is absorbed by the analyte. Transmittance is
calculated by dividing the transmitted light by the incident light (Equation 3).
Experimentally, however, transmittance is usually measured as the percent of the incident
light or %T and is defined as shown in Equation 4.
T = I/Io
%T = (I/Io) X100%
Absorbance is defined by Equation 5.
A = -log(T)
But since %T rather than T is actually measured, Equation 5 is changed to Equation 6.
A = -log(%T/100) = log(100/%T) = log 100 – log(%T) = 2-log(%T)
Each chemical species absorbs a different quantity of light at any given
wavelength. The amount of light absorbed by a compound depends on the structure of the
compound and the solvent. However, for any chemical species in a given solvent, the
amount of light absorbed at a given wavelength will be a constant called the molar
absorptivity, ε (also referred to as the molar extinction coefficient) (ε is the Greek letter
episilon) The absorbance, A, is a function of the concentration, c, of the absorbing
species and the distance the light travels through the solution, that is the pathlength, l.
Absorbance can be calculated for any solution for which the molar absorptivity is known
A = ε.c . l
Equation 7 is often referred to the Beers-Lambert Law or Beers’ Law for short.
Since molar absorptivity and pathlength are both constants for a given instrument, one
may safely assume that the absorbance is directly proportional only to the concentration
of the analyte. The concentration of a solution can be determined from a Beers’ Law
calibration plot prepared by measuring the %T (or A) for several solutions of known
concentration (standard solutions). The Beers’ Law plot uses concentration as the
independent variable (x-axis) and absorbance as the dependent variable (y-axis) as shown
in Figure 1. When the absorbance of an unknown sample is determined, the concentration
can then be determined by graphical interpolation from the prepared calibration graph as
shown in Figure 1 for a solution with an absorbance of 0.500.
Beer's Law calibration graph
y = -0.001 + 0.055125x R= 0.99997
Absorbance = 0.50
Concentration = 9.0 mg/mL
Figure 1. A Beers’ Law plot for an absorbing species with ε = 5.56 X 10-2 and l =
Figure 2 shows an overview of the Spectronic 20 (Spec 20) spectrometer that will be used
in this experiment. Figure 3 provides an optical diagram for the instrument.
Analysis of Brass
Brass is an alloy composed of zinc and copper. In order to determine the copper
content in a sample of brass we must first dissolve the sample. To achieve this we will
use nitric acid, a strong oxidizing acid. The reaction can be described by the following
3Cu(s) + 8H+(aq) + 2NO3-(aq) J 2NO(g) + 4H2O(l) + 3Cu+2(aq) Equation 8
The nitric oxide, NO, gas evolved in this reaction reacts immediately with oxygen to
form brown NO2 and N2O4 gases. NO, NO2, and N2O4 are all toxic. The dissolution
process therefore must be done in a working fume hood.
Figure 2: An overview of the Spec 20 Spectrometer
Figure 3. A schematic diagram of the optical diagram for a Spec 20.
Zinc will also dissolve in nitric acid according to the following equation.
Zn(s) + 2H+(aq) J Zn+2(aq) + H2(g)
Once the sample has been dissolved in nitric acid we need a sensitive test for copper (II)
ions in the presence of zinc (II) ions. A spectrophotometric test takes advantage of the
fact that copper solutions are blue while zinc ions are colorless. However, aqueous
copper (II) ions are light blue in color and the detection limit is quite low because the
molar absorptivity of aqueous copper (II) is small. To compensate for the low ε of
aqueous copper we will intensify the color by complexing it to ammonia according to
Cu+2(light blue-green) + 4NH3 J [Cu(NH3)4]+2 (deep navy blue)
Copper (II) ions readily form a tetraamine complex while zinc (II) does not.
Consequently, copper becomes readily detectable in a UV-VIS spectrometer while zinc
ions remain invisible.
In order to make a Beers’ law plot it is necessary to construct a standard curve.
This is most easily done by preparing a single standard solution with a known
concentration of copper then diluting the solution to several lesser concentrations.
Equation 11 is used to calculate the amount of known solution needed to prepare a
dilution of a known concentration.
C1V1 = C2V2
In this equation C2 and V2 are the known values of concentration and volume of the
desired solution respectively and C1 and V1 are the concentration and volume of the
known solution. For example, if I have a known solution containing 10.0 mg/mL (C1)
copper (II) and I want to make 50.0 mL (V2) of a solution containing 3.0 mg/mL (C2)
copper (II), I would use equation 11 as follows
(10.0 mg/mL)V1 = (3.0 mg/mL)(50.0 mL)
Then V1 = [(3.0 mg/mL)(50.0 mL)/(10mg/mL)] = 15.0 mL
Thus I need to dilute 15.0 mL of the 10.0 mg/mL solution to 50.0 mL to make the desired
3.0 mg/mL solution.
Safety: Note that the gases produced during the dissolution of the brass sample are toxic.
This step must be done in a fume hood.
1. Obtain a sample of brass. Record the actual mass of the sample which will be written
on the container.
2. Place the sample directly into a 50 mL volumetric flask containing approximately 5-7
mL of concentrated nitric acid (Do this in the fume hood). Record your observations
concerning the reaction.
3. While the sample is dissolving, prepare the assigned standard solution. The
concentration of the standard will be assigned to each pair by the instructor. The
standards should be in the range of 0.2 to 1.0 mg/mL. Each pair of students should
prepare a different standard solution from the stock solution which will be
approximately 10 mg/mL. The standards will be shared among all pairs of lab
students. To prepare the standard solution:
a. Calculate the volume of stock solution needed to prepare the standard with the
assigned concentration as described above. Note that the volume of standard
solution will depend on the volume of the volumetric flask that you will be
b. Drain the calculated volume (measured to +/- 0.2 mL) of stock solution from a
buret into a volumetric flask. Record the actual volume of the stock solution to
the nearest 0.1 mL. Calculate the accurate concentration of the standard in
mg/mL using the actual volume of stock solution drained.
c. Add about 10 mL of water to the flask. In the fume hood add concentrated
ammonia 1 mL at a time, swirling the flask after each addition. Continue the
additions until you obtain the deep blue color of the copper (II) tetraamine
complex. When the color doesn’t get any deeper, add an additional three mL
of ammonia. Fill the volumetric flask to the mark with distilled water.
Remember to read the meniscus at the bottom of the curve. Stopper the flask
and mix the solution well. Label the flask with the concentration (calculated to
the nearest 0.01 mg/mL).
d. Place the standard solution that you prepared with those of other students in a
place indicated by the instructor. Each pair of students should use at least six
of the standard solutions as outlined below to construct their experimental
Beer’s law calibration curve.
4. Next finish the preparation of the brass solution from step 2 above. Once the brass has
dissolved, add 10 mL of water then ammonia as described in step 3c above. Fill the
volumetric flask to the mark with distilled water. Stopper and mix well.
5. To perform the measurements using the spectrometer (see the appendix for additional
a. Set the wavelength to 610 nm (this is λmax for the Cu(NH3)4+2 ion).
b. Adjust the display to 0% by turning the power switch/Zero control knob on
the front of the instrument.
c. Fill a clean cuvette half-way with distilled water (this is the blank or reference
d. Wipe the outside of the cuvette with a paper towel to remove any fingerprints,
water, or dust particles. After cleaning, handle the cuvette only by the upper
rim. Place the filled cuvette into the sample compartment. Align the guide line
on the cuvette with the guide mark on the sample compartment.
e. Adjust the readout dial to 100%T with the Transmittance/Absorbance control
f. Remove the cuvette, empty it and rinse twice with a small amount of the first
standard solution to be measured. Add standard solution to fill the cuvette at
g. Wipe the outside of the cuvette and place it into the sample compartment,
aligning the guide line of the cuvette with the guide line of the sample
h. Read the value of %T (or absorbance) from the scale. To read the scale, make
sure to look at the dial in such a way that you see only a single line in the dual
i. Repeat steps f-h using a new standard solution each time. Measure at least 6
standard solutions. Finally, measure your unknown brass solution.
j. Remove and clean the cuvette. Turn off the spectrometer by turning the power
switch knob counterclockwise until it clicks.
Note: The lower concentration standards will require filtration if they contain a
white precipitate. To perform filtration, use a clean, dry funnel with a pre-
pleated filter paper. Filter the solution directly into the cuvette after prerinsing with a small amount of the solution. Use a separate clean and dry
funnel and filter paper for each solution.
6. Dispose of all solutions in the waste container provided. Clean all glassware used
for the experiment.
Calculations and Report
1. Show all calculations for the standard solution that you made.
2. Calculate the absorbance for each standard and the unknown brass sample.
3. Create the Beers’ law calibration graph. Use graph paper. Draw the best fit
(straight) line through the data points. If any of the data points appear to be far off
the straight line you may exclude it from the graph but the exclusion requires an
explanation in your discussion. You may use a computer to make your graph;
however, a copy of the graph must be included in your report. A computer graph
must include labeled axes (with appropriate scale marks) and the best fit line must
be fitted through the points.
4. Use the created calibration graph (or the equation of the best fit line) to determine
the concentration of copper in the solution.
5. Use the concentration of copper in the unknown brass solution and the volume of
the solution to calculate the mass of copper in the original brass sample.
6. Calculate the weight percent of copper in the brass sample. %Cu = (mass of
copper/mass of brass) X 100%.
Write a brief discussion of your results in which you state your experimental results
including the mass of copper in the sample and the % copper in the brass sample.
Describe any errors made and state ways you could improve your experimental
Experiment 12 Data Table
Mass of brass (samples are preweighed) ___________________________
Volume of sample created ________________________________
Chemistry 1215, Experiment #12; Spectrophotometric Analysis of an Unknown Brass
1. Explain why is it necessary to wipe the outside of the cuvette before performing a
2. Use the graph in Figure 1 to determine the concentration of an unknown sample for
which the measured %T is 20%. Show any calculations.
3. A student correctly prepared the standard solutions used to create a standard curve for
copper analysis. However, in preparing his brass sample he forgot to add ammonia.
Explain in detail what effect this error will have on his final result. Will the measured
percent copper in the brass sample be high or low?
4. A student used a 10.54 mg/ml copper stock solution to prepare a set of standards to
create a copper standard curve. If the standard solutions were made in 50.0 mL
volumetric flasks, how much of the stock solution should be diluted to make a final
concentration of 0.75 (mg Cu)/mL?
Chemistry 1215, Experiment #12; Spectrophotometric Analysis of an Unknown Brass
1. When determining the Transmittance of the brass sample solution, a student rinsed
the cuvette with water but not with sample solution before taking the measurement.
Explain in detail what effect of this error will have on the final result. Will the final
answer be high or low?
2. A 50 mg brass sample was placed in a 50.0 mL volumetric flask. The sample was
prepared according the procedure outlined in this lab and its transmittance was
measured to be 35%. Use the graph in Figure 1 to determine the mass percent of
copper in the sample. Show all calculations.
3. In an experiment a student used a 50 mL volumetric flask to prepare her brass
solution but in calculations she used 100 mL as the sample volume. What effect will
this error have on the final result? Will the calculated result be high of low?
4. Methyl viologen (also called paraquat) is a redox active dye that is blue in the
reduced state and colorless in the oxidized state. The reduced compound is capable of
passing electrons to and from the active centers of redox active enzymes. The molar
absorptivity of methyl viologen at a wavelength of 600 nm is 13,000 L/(cm.mol).Use
Beer’s law to determine the concentration of a methyl viologen solution that has an
absorbance of 1.600 in a 1.00 cm cuvette.
Appendix 1: Using a Spectronic 20 (From the Dartmouth University website)
A Spec 20 spectrometer measures the amount of visible
light absorbed by a colored solution. This can be read as
Absorbance or % Transmittance.
Setting Up the Spectrometer
Check that the instrument is turned on. The lefthand knob should be turned clockwise. Allow 10
minutes for warming up.
Set the wavelength to the desired value using
the knob on the top.
Calibrating the Spectrometer
With empty, closed sample compartment, turn the lefthand knob to obtain a reading of 0% T.
Use the mirror behind the needle to avoid
Use the arrow button below the image to view more about
parallax error. Having trouble viewing the slideshow? Help is
Wipe the cuvette with a dry Kimwipe to remove
drops of solution or finger prints.
Line up the mark on the cuvette with the line on
the sample compartment.
Insert cuvette filled with solvent in the sample
Close the cover.
Turn the right-hand knob to obtain a reading of 100% T.
Using the Spectrometer
To analyze your sample, insert sample cuvette and read
the Absorbance value on the scale. Use the mirror
behind the needle to avoid parallax error.