Experiment 10
Absorption Spectroscopy
In the first part of your experiment today, you will observe the H2 gas emission spectrum. The
remainder of your experiment will be carried out using an instrument called a Spectronic-20 (or
Spec-20, for short). First, you will examine what wavelengths of visible light correspond to various
colors of the spectrum. Then, you will see what happens to the light when it hits a colored
compound. Finally, you will examine how solution concentration is related to the absorption of light
and use this relationship to determine the concentration of an unknown.
10.1 Introduction
Light in the electromagnetic spectrum behaves much like waves and different types of radiation are
classified according to their wavelength (λ), frequency (ν) and energy (E). Some examples of types
of radiation are X-rays, UV light, infrared light, and microwaves. The type of light humans can see
is called visible light and the wavelength for this type of radiation goes from about 400 - 700 nm.
When energy is absorbed by an atom the energy is used to excite electrons to higher energy
level(s). When the electrons return to lower energy levels, the excess energy is released as
electromagnetic radiation. The energy of the emitted corresponds to the energy differences
between principle quantum levels for that atom. For example, when excited, hydrogen gas will glow
because it is emitting light in the visible region. The light emitted from hydrogen will always have
characteristic wavelengths or colors.
All kinds of radiation (or light) can interact with certain compounds in certain ways. For
example, all colored compounds absorb some wavelengths of visible light. The amount of light
that’s absorbed is determined by measuring the decrease in light intensity as light passes through a
sample. The percentage of the incident light intensity that is transmitted is reported as percent
transmittance (%T ) as shown in Figure 10.1.
Incident light
( intensity = I0 )
Transmitted light
( intensity = I )
%T=(I/I0)·100
0
Figure 10.1: Light intensity is reduced as it passes through a sample.
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Experiment 10
10.2
Experiment
Prepare your laboratory notebook.
10.2.1 Observation of the H2 Emission Spectrum
The hydrogen lamp at the front of the room consists of a glass tube filled with H2 gas. When
turned on, an electrical current passes through the gas, giving the hydrogen excess energy. This
excess energy is then emitted as light. Look at the hydrogen lamp using a spectroscope, a device
containing a grading which splits light into different wavelengths or colors (similar to how a prism
can spread white light out into a rainbow). Record your observations in your laboratory notebook
and include a sketch. (Don't forget to include a description of what it is you're doing!)
Calculate the wavelength (λ) in nm of the transition from the third energy level (n = 3) to the
second energy level (m = 2) using the Rydberg equation:
1
1
1
= 1.097 × 107 m-1 � 2 − 2 �
𝜆𝜆
𝑚𝑚
𝑛𝑛
10.2.2 Correlation of Wavelength and the Color of Light
You will use the reflection of light off a white piece of chalk to determine which wave­
lengths of light correspond to which colors.
1. Plug in the Spec-20, and turn the knob on the left-hand front of the instrument (ZERO
ADJUST) to the right; a click will signal that the instrument is turned on. Adjust the meter
reading needle all the way to the left mark (zero percent transmittance) using the ZERO
ADJUST knob.
2. Turn the light INTENSITY control knob (right-hand front) all the way to the right (clockwise).
3. Place a piece of chalk in a cuvet (the glassware that looks like a small test tube) and slide the
cuvet into the holder on the top left of the instrument. Do not put the chalk directly into the
instrument! Turn the cuvet until the flat side of the chalk is facing towards the right of the
instrument so that you will be able to see the reflected light.
4. Keep the cover of the cuvet holder open so that you can look down on the surface of the chalk.
Turn the INTENSITY selection knob on the top-right of the instrument all the way to the right to
be certain that you can see the bright light beam.
5. Set the WAVELENGTH knob to 400 nm and record the color of light reflected off the chalk for
each 25 nm step in wavelength up to 700 nm. Record your observations in a table in your
laboratory notebook. Use Table 10.2 as a guide for how to set up your table. You may not
see any color at the far ends of the spectrum.
Table 10.1: Sample data table.
Wavelength (nm) Color Observed
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Experiment 10
10.2.3 Absorption Spectrum
You will now monitor what happens to light intensity as certain wavelengths of light interact with two
different colored materials. In a table in your lab notebook, record the percent transmittance as a
function of wavelength for the red and the green dye. Record your results in a table similar to Table
10.2.
Table 10.2: Sample data table.
Wavelength (nm) %T for red dye %T for green dye
etc.
In each measurement, a blank (distilled water) is used to calibrate the Spec-20 at each wavelength.
1. Place the standard aqueous red dye solution into one cuvet (2/3 full), and the green dye
solution into a second cuvet (these are your SAMPLES). A third cuvet with distilled water is
your BLANK.
NOTE: Steps 2-4 must be repeated EACH TIME YOU CHANGE THE WAVELENGTH
2. . Set the wavelength (the first setting will be 400 nm). With no cuvet in the instrument and the
cover closed, use the ZERO ADJUST knob (the knob on the left) to set the meter reading
needle to the far left mark (zero percent transmittance).
3. Place the water BLANK cuvet in the holder; close the cover; and use the INTENSITY control
knob (the knob on the left) to set the meter reading needle to the far right reading (100%
transmittance).
4. You have now adjusted the instrument for this wavelength. DO NOT ADJUST THE KNOBS!
Place the red SAMPLE cuvet in the holder, close the cover and record the % Transmittance
(%T). DO NOT ADJUST THE KNOBS! Place the green SAMPLE cuvet in the holder, close
the cover and record the % Transmittance (%T).
5. Advance the wavelength to 425 nm and repeat steps 2-4. Continue advancing by 25 nm
increments and repeating steps 2-4 until 700 nm is reached.
6. Enter your data into an Excel spreadsheet. Calculate absorbance (A) from your %T data
using the formula A = 2 − log(%T ). (Your instructor can demonstrate how to do calculations
in Excel.) Then, construct a scatter plot (with lines connecting the data points this time!)
of absorbance (y-axis) vs. wavelength (x-axis). Overlay the results for both colored solutions
on the same graph. This type of plot is called an absorption spectrum. Since absorption is
based on a ratio of intensities, it is a unit-less value.
7. Type or hand-write the colors (red, green, etc.) you determined in the previous section under
their corresponding wavelengths on your graph.
8. Observe the solution of copper sulfate pentahydrate displayed at the front of the lab. Record
the color of this solution in your lab notebook.
Summary: Look at your data for the red and green dyes and your observations with the chalk. Can
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Experiment 10
you make any correlations between the color of a solution and the wavelength of maximum
absorption? Can you extrapolate this correlation and predict the wavelengths of maximum and
minimum absorption for the copper sulfate pentahydrate solution?
10.2.4 Calibration Curve
The amount of light absorbed by a sample depends on the absorbing compound present in the
sample. Therefore, absorbance can be used to determine the concentration of that compound
present in a sample. You only need to use the red dye for the remainder of the lab.
1. For this part of the experiment set the instrument wavelength to 525 nm. This is your
ANALYTICAL WAVELENGTH.
2. Use two 10 mL graduated cylinders (one for water and one for the red dye) and two disposable
pipets to prepare the following six solutions in six small beakers. Label each beaker as you
go. In your notebook, set up a table like the one below.
Table 10.3 Sample Data Table
Solution
Vol. of H2O (mL)
Vol. of red dye (mL)
1
5
0
2
4
1
3
3
2
4
2
3
5
1
4
6
0
5
Conc. of red dye (M) %T
3. Calculate the concentration of red dye in each solution using the formula C1V1 = C2V2 where C1
is the initial concentration of the standard solution, V1 is the volume of the standard solution,
C2 is the final concentration of the diluted sample, and V2 is the total volume of the diluted
solution.
4. Also in your notebook, predict which solution will absorb the most light and which solution will
absorb the least light? (Explain your reasoning.)
5. Calibrate the instrument using solution 1 as the BLANK at the analytical wavelength only.
(Without the cuvet, set the %T to zero, and with the cuvet in place, set the %T to 100%.) After
this calibration, you won’t need to touch the wavelength and intensity knobs again.
6. Empty the cuvet and rinse it with a small amount of the solution you are about to measure.
(Start with the lowest concentration solution). Place the cuvet with the sample in the
instrument, and record the %T. Pour each solution back into its original beaker when you’re
finished recording your data.
7. Enter your data into an Excel spreadsheet as before and calculate the absorbance for each
solution. Construct a scatter plot (with no connecting line this time!) of absorbance (y-axis)
vs. concentration (x-axis). Fit a trendline and obtain the equation for the line. This graph is a
Beer’s Law calibration curve and should be a straight line. Beer’s Law tells us that the
concentration of the red dye is proportional to the absorbance and can be used to determine
the concentration of solutions of unknown concentration.
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Experiment 10
8. After your graph has been constructed, your instructor will give you one solution red dye with
an unknown concentration. RECORD THE UNKNOWN NUMBER. Then, measure and
record the %T of the solution.
9. Convert the %T of your unknown solution to absorbance. Then, use the equation of your
Beer’s Law plot to calculate the concentration of red dye in your unknown sample.
10.3 Wrap Up
10.3.1 Waste Handling
The red and green dyes used in this experiment are the same dyes used in food coloring and
therefore are not toxic. All solutions should be poured down the sink.
10.3.2 Clean-up
DO NOT THROW AWAY YOUR CUVETS. Clean and dry your glassware and return it to your
station. Then, wipe down your bench top with a wet paper towel and dry it. Have your instructor
check your station before you leave.
10.5.3 Work Assignment
Enter all of your data, calculations and answers to questions in the Experiment 10 Assignment in
Chem21. You will need to refer to your notebook for this. The Assignment is due at 8 AM on the
day of your next lab meeting. In addition, to the Chem21 assignment, you will prepare 2 graphs.
One is the absorption spectrum of the red and green dyes (see 10.2.3 steps 6 & 7) and the other is
the Beer’s Law plot for the red dye (see 10.2.4 step 7.) Bring a hard copy of these graphs to your
next lab meeting.
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Experiment 10