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					Physics 598OS Optical Spectroscopy (Fall 06)                                                 Clegg/Chao/Kijac




                               Lab Writeup Instructions
  Due at your next lab section (Thur Sep7 or Fri Sep 8). Labs will generally be due the next week at your
section. For this time, if you are in a Thursday section you can turn it in on Friday if you want – you’ll just
                                     have to make the extra trip to Loomis.

The questions are pretty much what you got on Thursday for with your original lab writeup. I’ve included
the full revised writeup instead of a list of questions – it’s just clearer that way. Questions that you have to
answer have been clearly numbered and boxed off.
     • You can either write on this revised lab writeup directly, or on a separate sheet as long as
          questions are clearly numbered.
     • Tables and plots can be printed out and attached at the end of the lab report.
     • You only have to answer one of either Q3 or Q7, depending on which section of the lab you did
          first.
     • Q9: Change of plans: analyze the data I’ve taken and posted to get a quantitative spectrum. The
          original plan was to use the intensity of the water measurement at 490nm for as a rough Io for all
          wavelengths. But especially since we switched the light source to a flashlight so that the intensity
          light source itself already had a much stronger dependence on wavelength, this will not work.
          Since you did not take the “blank” measurements with water, use the TIF’s I’ve provided to
          analyze instead. You do not need to analyze or present the data you took for this question.
     • Your data is downloadable via the Labs section of the class webpage.
          http://online.physics.uiuc.edu/courses/phys598OS/fall06/

In general (i.e., except for Q9), for the Beckman DU experiment, if you did not get “good” results from
your data, you should still analyze/present your own data and give reasonable conjectures as to why your
data could be off. (Note, that the whole method is too crude is not an acceptable reason. I was able to
measure concentrations within 5% for solutions A and B using the Beckman.) If you want, you can feel
free to analyze and present another groups data as long as 1) you’ve analyzed and presented your own data
and 2) you clearly acknowledge where the data came from.

As for the monochromator concentration measurements, don’t worry too much about the actual
concentrations you measured. Just clearly present your data. Graduated ND filter calibrations are
presented at the end, along with descriptions so that you can figure out which one you used. For the lab
report, please indicate the filter you used following the naming convention in the appendix (i.e., A, D, E,
etc.) I will bring the filters to lecture on Tuesday so you can be sure of which filter you used.

The concentrations of the fluorescein solutions are:

                           Name             Concentration (µM)
                           A                                        7
                           B                                        3
                           C                                     0.35


Finally, the first thing you need to do as part of your lab writeup assignment is to set up your Active
Directory password. Go to the following site and follow the instructions:
http://www.ad.uiuc.edu/




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Physics 598OS Optical Spectroscopy (Fall 06)                                            Clegg/Chao/Kijac




                  Lab 1 – Monochrometers and such…
I. Introduction
This lab is exploratory and introductory. If you use absorption spectrometers and fluorimeters in your
everyday research, here’s your chance to get in under the hood. If you don’t, now’s your chance to get
acquainted with these instruments on a rudimentary level. You should approach this lab with an
exploratory, hands-on perspective. We will be doing some crude absorption measurements manually,
without the automation of modern instruments

                                       Topics Covered
                  •   Monochrometers
                  •   Wavelength Dispersion Elements
                          o Diffraction Gratings
                          o Prisms
                  •   Fluorescein – absorption/fluorescence



References
1) Jeremy M. Lerner, “Imaging spectrometer fundamentals for researchers in the biosciences - a tutorial”
        http://www.lightforminc.com/ImagingSpectrometerFundamentals.pdf
        Accepted for publication in the journal "Cytometry"
        (http://www3.interscience.wiley.com/cgi-bin/abstract/112593104/ABSTRACT)

2) The Instrument Project: UV-visible spectroscopy.
http://www.wooster.edu/chemistry/is/brubaker/uv/default.html

3) Eugene Hecht, Optics


II. Monochrometer Experiment
In this part of the lab you will be playing with a Bausch & Lomb grating monochromator (circa 1950s).
First, familiarize yourself with the operation of the monochrometer. Then you will use the monochrometer
to explore the qualitative absorption properties of a fluorescein, a highly absorbing and fluorescent
compound. Finally, you will perform a rudimentary absorption measurement by comparing the intensity of
the transmitted light to a known reference by eye.

                               • While these monochromators are old, the mirrors and
                                 diffraction gratings are valuable! Do not touch/clean
                                 the mirror or grating surfaces. Gratings are
                                 destroyed and mirror surfaces seriously damaged by
                                 fingerprints. They’ve survived this long, so give ‘em
                                 another 60 yrs.
                               • The filters used in this lab are Prof. Clegg’s research
                                 equipment. They are expensive! Do not touch
                                 optical surfaces and treat with extreme care.



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Physics 598OS Optical Spectroscopy (Fall 06)                                             Clegg/Chao/Kijac




     Monochromator Basics

There are two monochromators available for use. Monochromator A on the lab bench and Monochromator
B on the cart. Both can be used for Part IIA.

Familiarize yourself with the optical path through the spectrometer. The 100W desk lamp serves as your
light source. Follow the light path from the lamp through the entrance slit all the way to the output slit.

                                       Monochromator Schematic

Q1) Draw a top down 2-dimensional schematic of the path the light takes through the monochromator.
    • Include
       1) light source, 2) entrance/exit slits, 3) mirrors, 4) diffraction grating.
    • The schematic should be specific to this monochomator.
    • Clearly label all components




    •   What role do the entrance and exit slits play in selecting the wavelength? Do you want a
        wider/narrower slit for better wavelength selection?




Recall from the physics of diffraction gratings that for a particular wavelength λ, you can have multiple
maxima (one at each order k). These monochromators use a blazed diffraction grating consisting of
repeated triangular shaped rulings, separated by distance a and with a blazing angle ω. For your lab
writeup, you should make sure you understand why for a given λ there is a kth order maximum at angle ω
given by:




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Physics 598OS Optical Spectroscopy (Fall 06)                                                  Clegg/Chao/Kijac




                                                                              2a sin(ω ) = kλ




From Ref 1.
Note that this is for a special case where the incident angle equals the diffraction angle, the Littrow
configuration.

Look for the second order maxima in monochromator B, where the grating has been disengaged from it’s
turning knob, allowing you to turn the diffraction grating until the 2nd order maxima are visible.

Q2)    What if you wanted to use a grating monochromator to take a spectrum from 100 nm to 500 nm
       (e.g., the absorption spectrum of fluorescein, which you will be doing later in the lab). Say you scan
       to the first order maximum of 400 nm.
        a) Is that the only wavelength that would come out of the exit slit of the monochromator? Would
        you see other wavelengths? What order maxima would they be?




       b) How could you eliminate other wavelengths if they are present? (Hint: what kind of optical
       equipment could you use?)




 A. Using the Monochromator for Absorption Measurements
In this section, we use the monochromator to make some rough, rudimentary absorption measurements.
We will use fluorescein, a highly absorbing compound. The energy from any incident light which is
transferred to the molecule, sending it into an excited state. We get the absorption by measuring the
amount of transmitted light that is not absorbed (light that is scattered would also not be transmitted). In
lab 2, we will go into more detail regarding the theory behind absorption. For now, let’s just get down
some working “facts”.

Define the optical density of a solution by the log of the ratio of the incident and transmitted intensities
         OD = log( I 0 I )
         I = I 0 ⋅10 − OD
The optical density is composed of absorbing and scattering components
      OD = A + S

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Physics 598OS Optical Spectroscopy (Fall 06)                                              Clegg/Chao/Kijac


For this lab, we assume scattering is negligible, i.e., OD ~ A.

Intuitively, you would expect the absorption to                           sample
depend on the number of absorbing molecules
the light beam encounters, and hence on the                          C:   concentration
concentration of absorbers (C, mol/L), path
length through the sample (x, cm). So                  I0 (λ)                                  I (λ)
A = ε ⋅C ⋅ x                                          incident              extinction      transmitted
where ε, (L/mol-cm), the molar extinction intensity                ε(λ): coefficient          intensity
coefficient, is a fundamental property for each
compound. This is known as the Beer-Lambert                          x: path length
Law. For now, A~OD.
For fluorescein at 490 nm , ε =93000 M-1cm-1. x = 1 cm for the cuvettes we are using.


1) Crude Absorption Spectrum
N.B. If you started off with the Beckman DU and already drew a guestimated absorption spectrum, you
can skip this part. Although, it is still a good idea to quickly go through the motions to get a feel for the
instrument.

Scan the monochrometer through the visible spectrum to get an idea of the intensity of the lamp at each
wavelength. Using the fluorescein solution in the cuvette marked CONC (concentrated), again scan the
monochromator through the visible spectrum. Sketch a very rough absorption spectrum for fluorescein
(OD vs. λ) (Main feature to get, of any absorption peaks. I know this will be very rough, and the
monochrometer is also not calibrated. Just roughly use red 700 nm, green 550 nm and violet 400 nm as
landmarks.)

Q3)                   Rough Estimate Absorption Spectrum of Fluorescein (OD vs. λ)
You should give rough numbers for the λ axis. You don’t have to do that for the OD axis.




You will measure this more quantitatively later with the Beckman DU and a CCD camera.

2) Concentration Measurements
One of the uses of absorption measurements is determining solution concentrations. Here you will do this
by eye, matching the intensity transmitted by the fluorescein solution to that transmitted by a graduated
neutral density filter. Neutral density means it transmits (roughly) the same amount at all wavelengths.
Graduated means there is a spatial gradient in the OD across the filter. The graduated ND filter you are
given is linear, so given the distance d along the filter, you know OD(d). The procedure is as follows:




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Physics 598OS Optical Spectroscopy (Fall 06)                                                 Clegg/Chao/Kijac


•     The absorption peak of fluorescein is at 490nm. Since the monochromator is not calibrated, place the
      490 nm bandpass filter in front of the exit slit and adjust the monochromator wavelength until you see
      light. What wavelength should the light be at then?
•     Place the cuvette with fluorescein marked A into the clamp on the ring stand. Adjust the height of the
      cuvette so that it covers the top half of the exit slit. Place the graduated ND filter on the lab jack in
      front of the exit slit and adjust it’s height until it covers the bottom half of the exit slit.
•     Slide the filter back and forth until you find d for which the transmitted intensity matches that of the
      cuvette. Record d, as well as the uncertainty (cm) in your measurement (e.g., d = 5 ± 0.5 cm) .
•     Using the conversion table/equation provided (at end), convert your measured d into an OD. Then
      calculate the concentration of the fluorescein solution.
•     Hard to judge, isn’t it? Everyone in your group should make an independent measurement. There are
      three samples. If you have a large group, have different people measure different samples, but make
      sure each sample has more than one independent measurement so you can take the mean and std dev.


Q4)     a) For your own measurement, calculate the OD, and convert the uncertainty in d to an uncertainty
        in OD.




        b) Record all measured d for your group for each sample measured. Calculate the mean OD and
        standard deviation. Compare this value to the actual concentration which will be given to you after
        lab.




        c) Do a propagation of error calculation.
                 i) If you had a 10% error in measuring d, what is the error in your calculated concentration.




                 ii) With this experiment setup, it comes out that you basically write down an OD directly,
                 although you are really making a comparative judgment of the intensity of the transmitted
                 light. What if you were measuring an intensity directly (i.e., you were counting photons)
                 with a photodetector. In that case, if you had a 10% error in I, what error does that translate
                 to in your measured concentration? Assume Io is known exactly.




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Physics 598OS Optical Spectroscopy (Fall 06)                                               Clegg/Chao/Kijac



 Q4)       d) How does the graduated ND filter work? Hint: look at it on it’s side. If we use the Beer-
cont’d     Lambert law (which actually applies for solutions) to this situation, what parameter are we
           changing to change the OD as we slide along on the graduate ND filter (i.e., as d changes)?




           e) How can you improve this measurement? Obviously, using a photodetector to measure the
           transmitted intensity will much improve things. But what if you still used your eye. What would
           you tweak about this part of the lab to make it easier to measure the right concentration? What
           parameters can you tweak in the Beer-Lambert Law: (Io, C, x, etc., ) that would make it easier to
           differentiate between intensities by eye? What techniques/apparatus would you change?




3) Mystery Light?
Before moving on, keep the setup you have above, with the graduated ND filter matched to the intensity of
the transmitted light. Now place the 530 nm band pass filter in front of both the cuvette and the filter. You
should see light transmitted through the band pass filter from the cuvette, but not “from” the graduated ND
filter. This may be faint and difficult to see – you might try different concentrations if you don’t see it.

Q5)      How do you explain this light at 530 nm? Hints: What wavelength of light is coming from the
         monochromator and incident on the cuvette/graduated ND filter. How does the energy of the
         incident light compare to that at 530 nm? What could’ve happened to energy of the light absorbed
         by the fluorescein molecules? If you did not see this, answer this question (a) assuming that you did
         see it, and (b) suggest ways to improve the experimental setup so that you can see it (e.g., what
         could you change, Io, C; what filters could you use, etc.).




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    Physics 598OS Optical Spectroscopy (Fall 06)                                             Clegg/Chao/Kijac


    III. Beckman DU Experiment
    The Beckman DU uvVis-spectrophotometer is a landmark instrument that revolutionized chemistry,
    bioscience and medicine. (This is the same Beckman as the Beckman Institute.) Its design was motivated
    by the desire to use absorption spectra as “molecular fingerprints”. It was the first instrument to give
    reliable UV measurements and to combine optics with useful electronic instrumentation. Unlike the grating
    monochromator, the Beckman DU used a quartz prism as the wave dispersion element instead of a
    diffraction grating.



    A. Beckman-DU Basics

    Familiarize yourself with the optical path through the spectrometer. There are manuals and Beckman’s
    original paper at the bench.

    Go to the following website, and go through the tutorial illustrating the optics of the Beckman DU
    http://www.wooster.edu/chemistry/is/brubaker/uv/uv_works_du.html


                                           Beckman DU Schematic
Q6) Draw a top down 2-dimensional schematic of the path the light takes through the Beckman DU.
 Include
1) light source, 2) entrance/exit slits, 3) mirrors, 4) prism, 5) sample chamber
2) You can omit the electronics.
Clearly label all components




Pay particular attention to the wavelength dial. How does the change in wavelength per rotation depend on
the wavelength. (I.e., do you have to make more turns to change the wavelength in the UV or the
infrared?)




    Version revised for writeup.                       -8-
      Physics 598OS Optical Spectroscopy (Fall 06)                                              Clegg/Chao/Kijac


      B. Quantitative Absorption Measurements
      If your group started with the Beckman first instead of the monochromator, read the intro to section II. B.
      for the necessary absorption measurement equations.

               1) Crude Absorption Spectrum
      N.B. If you started off with the monochromator and already drew a guestimated absorption spectrum, you
      can skip this part. Although, it is still a good idea to quickly go through the motions to get a feel for the
      instrument.

      Scan the Beckman through the visible spectrum to get an idea of the intensity of the lamp at each
      wavelength. Using the fluorescein solution in the cuvette marked CONC (concentrated), again scan the
      monochromator through the visible spectrum. Sketch a very rough absorption spectrum for fluorescein
      (OD vs. λ) (Main feature to get: any absorption peaks. I know this will be very rough.)

Q7)             Rough Estimate Absorption Spectrum of Fluorescein (OD vs. λ)




      You will measure this more quantitatively later with a CCD camera.


      2) Concentration Measurements
      We will be using the CCD camera to measure the intensity of the transmitted light. The procedure will be
      as follows:
      • Use PixeLink Capture to save a TIFF image of the transmitted light.
      • Measure a mean intensity of the transmitted light from the saved TIFF file using the image analysis
           program NIH ImageJ (which is free to download (http://rsb.info.nih.gov/ij/)- if time becomes tight, you
           may need to analyze the images later with this program on your own computer).

      Ask your TA to walk you through the use of the programs. When taking images with Capture, you should
      make sure the images are not overexposed (that is the recorded image is not overly bright and saturated).
      Do this by changing the exposure time rather than the gain. Again ask your TA.

      •   Take a picture (and save it on your computer) for each of the four “samples”: air, empty cuvette, water,
          fluorescein cuvette A, fluorescein cuvette B. There is a slideable cuvette holder that you can quickly
          change between samples, but you will need to swap in the last cuvette.
      •   Use ImageJ to record a mean intensity of the transmitted light for each of the five samples. Save the
          resulting measurement table.




      Version revised for writeup.                        -9-
Physics 598OS Optical Spectroscopy (Fall 06)                                          Clegg/Chao/Kijac



Q8)   a)
      •    Present your data.
               o TIFF files should be available for your TA. Let him/her know where you’ve saved the
                    files at the end of your time with the Beckman DU so they can be transferred over to
                    the class server for future reference.
               o The measurement table from your intensity analysis in ImageJ, with each measurement
                    clearly labeled.
      •    Does the relative transmitted intensities for each sample make sense compared to the others?
           The empty cuvette shows a measured OD. Is this absorption, or something else? How does it
           compare to the cuvette of water?




      b) Calculate the concentration of your two fluorescein samples and compare with the given values.
      Use the intensity measured for water as your Io. Why should it make sense to “subtract out” the
      water ?




3) Quantitative Absorption Spectra (Instructions and Questions)
    • Pick 5 or 10 (as time allows) roughly equally spaced points in the visible spectrum and measure
        the intensity for a fluorescein solution using the CCD. If time is tight, ask your TA for
        instructions
             o Be sure to include 490 nm and 514 nm.
    • Extract the mean intensities from your snapshots using ImageJ as you did in the last section. Have
        the TA put your TIFF files on the server, and include the table of measured intensities from
        ImageJ.




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Physics 598OS Optical Spectroscopy (Fall 06)                                                          Clegg/Chao/Kijac



Q9)   Note: You were not told to take a “blank” measurement with water at each wavelength for Io. The
      plan was to use the measurement for water at 490nm from the previous section as a approximate
      stand-in, since the absorption of water does not change much with wavelength in this region.
      However, since we wound up using a flashlight as a stand-in light source (after the tungsten lamp of
      the Beckman gave out during lab), which has a much stronger dependence of intensity on
      wavelength, this does not work. So use the data provided instead to calculate the absorption
      spectrum. There is a TIF for solution B and water as well at each wavelength. See:
      http://online.physics.uiuc.edu/courses/phys598OS/fall06/Labs/LabData/Lab01/AbsSpectWBlank.zip

      a) Present your table of measured mean intensities, including any exposure time data and the
      normalized intensities. Exposure times were changed, so remember to divide the Mean by the
      exposure time for each measurement.




      b) Plot the quantitative, normalized absorption spectrum.
          • Calculate the OD for each wavelength measured. Use the water measurement at each
               wavelength as your Io. Make sure you normalize for any different exposure times.
               Normalize your absorption spectrum by the OD at 490 nm for plotting.
          • Compare this to the actual spectrum for fluorescein F1300. Download the this spectrum in
               a comma delimited text file at
               http://probes.invitrogen.com/servlets/spectra?fileid=1300naoh
               (See the “View data points for this spectra” link at the bottom of the page)
               Again normalize the absorption spectrum by the value at the 490nm peak. Place both your
               measured spectrum and the downloaded spectrum on the same plot, print out and attach to
               your lab writeup.
          c) Given that you understand the point of section II.B.3, why don’t you see a peak at 514 nm
               here?




Version revised for writeup.                             - 11 -
Physics 598OS Optical Spectroscopy (Fall 06)                                        Clegg/Chao/Kijac




                                                   The Beckman DU ushered science into a George
                                                   Jetson era. [From the Beckman Model DU
                                                   Spectrophotometer Instruction Manual (305-A),
                                                   Beckman Instruments Inc., (Fullerton, CA).]




IV. Synthesis Questions
You should look over these questions and think about them before you leave lab, so you can check over
things that you might need.

The Beckman DU and the grating monochromator used different wavelength dispersion elements.
Q10)    a) What are some advantages and disadvantages of each? E.g., you can think about how a
        diffraction grating works and also the wavelength/turn ratio for the prism on the Beckman DU.
        Which wavelengths are each suited for?




         b) The B&L Monochromators were optimized for infrared. What component determines which
         wavelength the monochromator is optimized for, and which parameter of that component do you
         tweak to do this. What if you wanted to design a UV grating monochromator? What would be
         the drawbacks in terms of resolution and dimensions? Keep in mind how you are physically
         dispersing the different wavelengths.




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Physics 598OS Optical Spectroscopy (Fall 06)                                      Clegg/Chao/Kijac


         c) The Beckman-DU was the first to give reliable UV measurements. But the first prototype,
         which used a glass prism, didn’t work in the UV. Why?




Version revised for writeup.                   - 13 -
Physics 598OS Optical Spectroscopy (Fall 06)                                              Clegg/Chao/Kijac




Appendix A – Graduated ND Filter Calibrations
Please state which filter you used according to the letter name below (A, D, or E)

d = 0 on the lighter end of the filter.
All filters are 22.5 cm long.




                                                          Scratched markings 3-002C on one side
                                                          1092 on the other.

Labeled 1.70D
Had tape with red ruler markings




Thinnest filter used with small piece of tape on one
end and scratch markings: 156 and 3-002A.

As for as I know, these are the filters each group used

Thursday 1-4PM: (Group A: Filters D & E), (Group B: Filter E)
Thursday 5-8PM: (Group A: Filter D), (Group B: ?)

I will bring the filters to lecture on Tuesday so you can make sure which one you used.




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