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PHYS 3153 Methods of Experimental Physics II
O4. Applications of Optical Spectroscopy
Purpose
In this experiment, you will study the principles and applications of optical spectroscopy.
Equipment and components
Spectrophotometer Kit (including Optics Bench, Degree Plate with Light Sensor Arm,
Collimating Slits and Lens, Grating (~600 lines per mm) with Mount, and Focusing Lens),
High Sensitivity Light Sensor (CI-6604), Rotary Motion Sensor (CI-6538), Aperture Bracket
(OS-8534), Mercury Vapor Light Source, Hydrogen Light Source, Incandescent Light
Source, Bengal Rose liquid sample and cuvettes (x6).
Background
A spectrophotometer is a simple scientific instrument, which allows you to view and measure
the spectral content (spectrum) produced by a light source. Figure 1 shows the optical
arrangement of a grating spectrometer. The collimating slit and collimating lens produce a
narrow beam of parallel light rays. The grating disperses the beam of light into a spectrum
with different wavelengths at different angles but with all of the light of a given wavelength
in a parallel beam. The focusing lens focuses these parallel beams into spectral lines. Figure
2 shows a spectrophotometer system to be used in the experiment. The narrow slit on the
aperture disk (part of the aperture bracket) allows light of a single wavelength to enter the
high sensitivity light sensor. The light sensor measures the intensity of the light while the
rotary motion sensor measures the angle to which the light is diffracted by the grating. You
can find the wavelength of light using the measured angle and the grating spacing d,
m λ= d sin θ, m = 0, 1, 2…
where d is the distance between the rulings on the grating, m is the order of the particular
principal maximum, θ is the angle of the diffracted light, and λ is the wavelength of the
incident light.
Figure 1 Grating Spectrometer
The grating disperses the beam of light into a first order spectrum and higher order spectra.
The higher order spectra are broader and less bright than the first order spectrum, and may
overlap. The Grating is blazed, so that one side of the spectrum is much brighter than the
other side.
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Figure 2 Spectrophotometer System (Top View)
Procedure
Exercise 1: Emission (Bright Line) Spectrum. Unlike an incandescent source, such as a
hot solid metal filament, which produces a continuous spectrum of wavelengths, light
produced by an electric discharge in a rarefied gas of a single element contains a limited
number of discrete wavelengths - an emission or “bright line” spectrum. The pattern of colors
in an emission spectrum is characteristic of the element. The individual colors appear in the
shape of “bright lines” because the light that is separated into the spectrum usually passes
through a narrow slit illuminated by the light source. Figure 3 shows a ray diagram for the
first order diffraction pattern of a multi-slit grating. The path length for Ray A is one
wavelength longer than the path length of Ray B. These two rays are in phase and an image
of the light source can be formed at this angle of diffraction.
Figure 3 Ray diagram for first order diffraction pattern
The purpose of this exercise is to determine the emission spectrum of mercury and hydrogen
vapor lights. The high sensitivity light sensor measures the relative intensity of colors of
light in an emission spectrum produced by a vapor light source passing through a grating. The
rotary motion sensor measures the angle, θ, of each band or “bright line” of color.
Equipment Setup
1. Set up the spectrophotometer next to a mercury vapor light source as shown in Figure 4.
If needed, adjust the lab jacks to raise the spectrophotometer to the same level as the
opening to the light source.
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2. Mount the grating on the grating mount. Caution: Handle the grating carefully. Avoid
touching the surface of the grating or glass plate to which the grating is attached.
Hold the grating through the edges of the glass plate.
3. Turn on the light source. Mask the opening of the light source, so that it transmits a beam
of width ~2cm to the collimating slits. Adjust the collimating slit slide so that the number
1 slit (the smallest one) is in line with the light source.
Figure 4 Equipment Setup
4. Once the light source is warmed up, adjust the light source, collimating slits, collimating
lens, and focusing lens so that clear images of the central ray and the first order spectral
lines appear on the aperture disk and aperture screen in front of the high sensitivity light
sensor. Turn the aperture disk so that the slit 2 (its width is 0.2mm) on the disk is in line
with the central ray.
5. Connect the Science Workshop interface to the computer and turn on the interface.
6. Connect the high sensitivity light sensor cable to Analog Channel A. Connect the rotary
motion sensor cables to Digital Channels 1 and 2.
7. Open the Capstone program “O4 Spectrum”. This program records and displays the
measured light intensity and the angle. You may use the built-in data analysis tools of the
program to find the angle for each color and then determine the corresponding
wavelength.
The following parameters are selected for this experiment (Refer to the Appendix 1 for
details on how to make these selections):
• Sample rate: 20Hz (20 measurements per second)
• Resolution of the rotary motion sensor: 1440 divisions per rotation
The actual angular position is calculated based on the angular position measurement
made by the rotary motion sensor and the ratio of the radius of the degree plate of the
spectro-photometer to the radius of the small post on the pinion. This is done using the
calculator function, as shown in Fig. 5.
In the program, a Graph Display Window is selected. The Light Intensity is on the
vertical axis and the Actual Angular Position is on the horizontal axis.
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Figure 5 Calculation of Actual Angular Position
Record Data
A. Spectrum scan
1. Darken the experimental area. Examine the spectrum closely. Determine which of the two
first order spectral patterns is brighter. Record your observation in your lab notebook.
2. Rotate the light sensor arm on the spectrophotometer to turn the degree plate until the
light sensor is beyond the last line of the brighter first order spectrum.
3. Set the GAIN select switch on top of the high sensitivity light sensor to 1.
4. Start recording data by clicking the “Record” button in the Controls Palette.
5. Push on the threaded post under the light sensor slowly and continuously to scan the
spectrum in one direction. As indicated in Fig. 6, scan the spectrum from one side of the
central ray all the way to the other side of the central ray.
6. Stop recording data by clicking the “Stop” button.
Figure 6 Scanning of the Whole Spectrum
7. Repeat the data collection procedures with the GAIN select switch set, respectively, to 10
and 100.
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B. Effects of the slit width
In part A, when you examine the “yellow line” of the mercury spectrum, you may not
notice that this “yellow line” is actually composed of two very close “yellow lines” (i.e. a
doublet with wavelengths of 577.0 and 579.1nm). In this part, this doublet will be
examined in details.
1. Examine the brighter second-order yellow lines with your eye.
2. Rotate the light sensor arm on the spectrophotometer until it is just beyond the yellow
lines.
3. Turn the aperture disk so that the slit 2 (its width is 0.2mm) is used.
4. Set the GAIN select switch to 100.
5. Start recording data. Then, push the threaded post under the light sensor slowly and
continuously in one direction to scan the yellow lines until the sensor passes through the
adjacent green line.
6. Stop recording data.
7. Repeat the data collection procedures with different input slit widths, by turning the
aperture disk to the slit 4, 3 and 1 (with slit width being 0.4, 0.3 and 0.1mm, respectively).
C. Spectrum of a different light source
1. Replace the mercury light source with a hydrogen light source. Caution: the mercury
light source is HOT.
2. Remove the collimating slits because the hydrogen light source is narrow enough.
NOTE: The hydrogen lamp has a finite lifetime and should be in operation only when
needed. Switch the lamp off when it is idle.
3. Obtain the first-order emission spectrum of the hydrogen light source with the following
settings:
Gain of light sensor: 100;
Aperture disk: slit 2 (0.2mm width).
Analyze Data
1. Use the Graph Display to examine the plot of Light Intensity versus Actual Angular
Position for the first set of data (GAIN = 1).
2. Use the built-in analysis tools to determine the angle of the first line in the first-order
spectrum and the angle of the matching line on the other side of the central ray.
3. Compute the difference between the two angles and use one-half of the value as the angle,
θ. Determine the wavelength, λ, of the first emission line.
4. Repeat the process for the other emission lines in the first-order spectrum.
5. Examine the plot of Light Intensity versus Actual Angular Position for the other two sets
of data (GAIN = 10 and 100). Look for the emission lines in the first-order spectrum that
may be too dim to detect when the sensor gain was set to GAIN = 1.
6. Record your data and generate a data table in your lab notebook like the one shown
below:
Color θ 1 θ 2 ∆θ θ = ∆θ/2 λ = d sin θ
7. Repeat the above analysis for the hydrogen spectrum.
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9. Examine the grating you used under a stereoscope on the side bench. Measure the grating
spacing d. Make sure that the stereoscope is properly focused so that you can view the
parallel grooves on the grating clearly. Note that the length of each scale in the eyepiece is
1cm (marked from 0 to 100). The actual size of an object under the microscope is given
by:
Actual size = Size on the scale / Magnification of the objective
Questions
1. Compare your results for the wavelength of colors in the mercury and hydrogen vapor
light spectra to the accepted values. Calculate the relative error of your measurements.
2. To accurately determine the wavelength, do you need a grating with a larger value of d or
a smaller value of d? Why? Give your explanations based on the grating equation.
3. Discuss the effect of changing the width of the input slit. What are the advantages and
disadvantages of using a narrower input slit.
Exercise 2: Absorption Spectrum. One of the most important applications of optical
spectroscopy is to identify substances by their absorption spectra. For example, it is possible
to identify tiny amounts of sodium dissolved in a complex fluid (such as beer), because
sodium has a unique absorption spectrum. An incandescent light source is used to produce a
continuous spectrum of wavelengths. A substance placed in the path of light will absorb the
light of certain wavelengths from the continuous spectrum. The individual wavelengths that
are absorbed appear as “dark lines” in the otherwise continuous spectrum (see Fig. 7).
Figure 7 Continuous spectrum and absorption spectrum
The purpose of this exercise is to determine the absorption spectrum of a liquid sample. The
high sensitivity light sensor first measures the continuous spectrum of an incandescent light
source and then measures the absorption spectrum produced when the incandescent light
passes through the liquid sample. The rotary motion sensor measures the angle, θ, of each
part of the spectra.
Equipment Setup
1. Set up the Spectrophotometer next to an incandescent light source as shown in Fig. 8. Move
the high sensitivity light sensor to the second position on the light sensor arm, so that you can
put a cuvette in between the back of the aperture disk and the opening to the light sensor.
2. Adjust the slide of the collimating slits so that the number 3 slit is in line with the light
source.
3. Turn on the light source. Once it is warmed up, adjust the light source, collimating slit,
collimating lens, and focusing lens so that clear images of the central ray and the first order
spectral pattern appear on the aperture disk and aperture screen. Turn the aperture disk so the
slit 2 on the disk is in line with the central ray.
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4. Follow the steps 5 to 7 in the Equipment Setup section of Exercise 1. Change the sample rate
to 50Hz (50 measurements per second)
Figure 8 Equipment Setup for Absorption Spectrum
Sample Preparation
In this part of the experiment, you need to measure the absorption spectrum of the Rose
Bengal solution at different concentrations. Rose Bengal is an organic dye. The master
aqueous solution of molar concentration ~6.3x10-5 M (moles/l) is stored in a storage vial.
Use a plastic pipette (see Appendix 2 for the Instructions for Use) to transfer the master
solution from the storage vial to 5 cuvettes and add distilled water to make five different
dilute solutions (It is recommended to prepare each solution with a final volume = 3ml).
Note: Use different pipettes for the master solution and distilled water. You may need 2
samples in the 10-5 M range and 3 samples in the 10-6 M range. Cover all the cuvettes with
plastic film after the solutions are made.
Record Data
1. Fill a cuvette with distilled water and cover the cuvette with plastic film. This is your
reference sample.
2. Put this reference sample in between the light sensor and the aperture disk. Make sure
that the smooth sides of the cuvette are in line with the opening to the light sensor.
3. Set the GAIN select switch on the top of the high sensitivity light sensor to 100.
4. Start recording data by clicking the “Record” button in the Controls Palette.
5. Push on the threaded post under the light sensor slowly and continuously to scan the
spectrum in one direction. As indicated in Fig. 9, scan the brighter side of the spectrum
only, but you have to scan across the zeroth order for analysis. HINT: The scan for each
sample should take about one minute in order to make sure that there are no missing data
points for comparison.
6. Stop recording data by clicking the “Stop” botton.
7. Repeat the above procedures for the five Rose Bengal samples.
Figure 9 Scanning the brighter side of the spectrum
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Analyze Data
1. Export all your data runs of the Light Intensity versus Actual Angular Position (five Rose
Bengal samples together with the reference sample). Select Export Data from the File
menu and then choose the data in the Export Data window, the data set(s) will be shown
as column(s) in table form.
2. Click the “Export to file…” button at the right bottom corner. Navigate to the desired folder and
type a name. Click “Save”. Then the data will be saved in a text file (*.txt) for a later use.
3. In the six data sets, it may occur that there are several same values of Actual Angular
Position data with different values of Light Intensity. You need to consolidate these data
before plotting the graph, please refer to Appendix 3 “Consolidate data using Microsoft
Excel” for details.
4. Use the graphic software Excel/Origin to plot the transmittance T versus wavelength for
samples of different concentrations. The transmittance T is defined as T = I/I0, where I is
the light intensity transmitted through the sample at a given concentration and I0 is the
light intensity transmitted through the reference sample.
5. The absorbance A is defined as A = -log10 (T). Plot the absorbance A versus wavelength
for samples with different concentrations. Find the maximum absorbance of the main
peak and plot it as a function of concentration. Can your data be fitted to a straight line?
Find a best-fit straight line, which goes through your data points.
Questions
1. What color corresponds to each of the “dark lines” in your absorption spectrum?
2. How does the absorption affect the apparent color of your liquid sample?
3. Beer's Law states that the absorbance of a solution is usually proportional to the
concentration, C, of the absorber in that solution, i.e., A = K C where K is a constant. Do
your samples obey Beer's Law? What is the value of K obtained from your
measurements?
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Appendix 1: Quick Reference for Capstone
Experiment Setup Window:
To setup a sensor and its properties options, click the “Hardware Setup” button on the Tools
Palette (as shown in Figure A1).
Figure A1 Capstone Experiment Setup Window
The sample rate can be changed on the sample rate setting on the Controls Palette (as shown
in Figure A1).
Graph Display Window:
The Graph Display window shows data sets plotted on an XY graph. A single Graph display
window can show multiple plots.
Figure A2 Graph Display Window
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Appendix 2: Instructions for Use of Digital micro Pipette
1. Volume setting
a. Turn the lock lever in the unlocking direction to loosen it. (Fig. A3)
Figure A3 Top view of the Digital micro Pipette
b. Turn the push button to set the digital counter to a desired liquid volume. Make sure
that the counter’s graduation is set at the point mark (red) as shown in Fig. A4.
Note: To avoid mechanical backlash, always finish setting clockwise. Increase the
volume setting by rotating the push button about 1/2 of a turn above the desired
setting, and then slowly turn back to decrease the volume until you reach the desired
setting.
Figure A4 Volume Indicator Figure A5 Side view of Digital micro
Pipette
c. After setting the liquid volume, turn the lock lever in the locking direction to lock it.
(Fig. A3)
Important: Do not exceed the specified liquid volume limit (1000µl), otherwise the
instrument will be damaged.
2. Extracting and Discharging Liquid
a. Press down the push button from point “a” to the first stop position “b”. (Fig. A5)
Note: Do not extract the liquid with the push button pressed at the second stop “c”.
b. Hold the pipette vertically and immerse the tip 2mm to 3mm below the surface of the
liquid. (Fig. A6-○1 E A)
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c. Release the push button slowly and smoothly to extract the set volume of the liquid.
Keep the pipette tip stationary for about 1 second to wait until the liquid is completely
sucked into the tip. (Fig. A6-A○2E A)
Note: Be careful to operate the push button very gently. If it is rapidly released, the
liquid may possibly be sucked into the main body and pipetting may end with an
inaccurate result.
d. Always dispense with the pipette tip against the wall of the container and pipette held
at about 45º to the wall. (Fig. A6-A○3E A)
Figure A6 Extracting and Discharging Liquid
e. Press down the push button slowly and smoothly to the first stop "b" as shown in Fig.
A5. Wait for a few seconds then press down the push button to the second stop point
"c" to expel the last drop of the liquid from the tip. (Fig. A6-A○4E A, A○5E A)
f. To get a rough calibration, you can pipette distilled water onto a container on an
electronic balance. Since the density of water is 1g/ml, 1,000 µl of distilled water
should weigh 1.0 gram.
g. Always keep the pipette in upright position during use and storage – do not allow any
liquid flow into the body of the pipette.
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Appendix 3: Consolidate data using Microsoft Excel
1. Open your data using Microsoft Excel.
2. Click on an empty cell (C1). Then, click on the Data tab and select Consolidate as shown in Fig.
A7.
Figure A7 Consolidate data
3. A Consolidate Window pops up. Choose:
Function -> Average; Reference -> $A:$B;
Check the checkbox “Use labels in Left column”;
and Click OK.
Figure A8 Consolidate Window
4. Consolidated data appears in column C and column D.
Figure A9 Consolidated Data