Applications of Optical Spectroscopy

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.

Revised: 20 August 2014 O4-1/13

Applications of Optical Spectroscopy

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.



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 DataStudio 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.



Figure 5 Calculation of Actual Angular Position 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.

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 “Start” icon in the program. 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” icon.

Figure 6 Scanning of the Whole Spectrum 7. Repeat the data collection procedures with the GAIN select switch set, respectively, to 10

and 100.



B. Effects of the slit width 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. 8. 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 give 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.

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 “Start” icon in the program. 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 two minutes in order to make sure that there are no missing data points for comparison.

6. Stop recording data by clicking the “Stop” icon. 7. Repeat the above procedures for the five Rose Bengal samples.

Figure 9 Scanning the brighter side of the spectrum



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 measurement, you can choose the “Light intensity vs Actual Angular Position” to export all the data runs or particular run if you want.

2. Use the Save File window to locate where you want to save the file. Type in a filename and click Save. Check the corresponding text files for your data, make sure the data are reasonable for further analysis.

3. In the data files, 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?



Appendix 1: Quick Reference for DataStudio Experiment Setup Window: To setup a sensor and its properties or sampling options, click the “Setup” button and the Experiment Setup window appears (see Figure A1 below).

Figure A1 DataStudio Experiment Setup Window Sensor Properties Window: To view or setup sensor properties, such as sample rate, measurement, resolution of the sensor, double-click the sensor icon and the Sensor Properties window appears (see Figure A2 below).

Figure A2 Sensor Properites Window



Graph Display Window: The Graph display window shows data sets plotted on an XY graph. A single Graph display window can show multiple plots. For example, you can use the Graph display window to show position versus time data, velocity versus time data, and/or acceleration versus time data for a mass oscillating on a spring or a freely falling object.

Figure A3 Graphic Display Window



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. A4)

Figure A4 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. A5. 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 A5 Volume Indicator Figure A6 Side view of Digital micro Pipette c. After setting the liquid volume, turn the lock lever in the locking direction to lock it.

(Fig. A4) 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. A6)

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. A7-○1E A)



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. A7-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. A7-A○3E A)

Figure A7 Extracting and Discharging Liquid

e. Press down the push button slowly and smoothly to the first stop "b" as shown in Fig. A6. 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. A7-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.



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.

A8.

Figure A8 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 A9 Consolidate Window 4. Consolidated data appears in column C and column D.

Figure A10 Consolidated Data


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Applications of Optical Spectroscopy