Optical Spectroscopy Lab Notes: Physics 598OS (Fall 06), Lab Reports of Physics

Download Optical Spectroscopy Lab Notes: Physics 598OS (Fall 06) and more Lab Reports Physics in PDF only on Docsity! Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Kijac Version revised for writeup. - 1 - 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/ Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Kijac Version revised for writeup. - 2 - 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. Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Kijac Version revised for writeup. - 5 - For this lab, we assume scattering is negligible, i.e., OD ~ A. Intuitively, you would expect the absorption to depend on the number of absorbing molecules the light beam encounters, and hence on the concentration of absorbers (C, mol/L), path length through the sample (x, cm). So xCA ⋅⋅= ε where ε, (L/mol-cm), the molar extinction coefficient, is a fundamental property for each compound. This is known as the Beer-Lambert Law. For now, A~OD. ε(λ): extinctioncoefficient concentrationC: incident intensity I0 (λ) transmitted intensity I (λ) x: path length sample 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: Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Kijac Version revised for writeup. - 6 - • 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. Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Kijac Version revised for writeup. - 7 - Q4) cont’d d) How does the graduated ND filter work? Hint: look at it on it’s side. If we use the Beer- 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.). Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Kijac Version revised for writeup. - 10 - 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. Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Kijac Version revised for writeup. - 11 - 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? Physics 598OS Optical Spectroscopy (Fall 06) Clegg/Chao/Kijac Version revised for writeup. - 12 - 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.