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Lab Writeup Instructions
Due at the beginning of your next lab section. Labs will generally be due the next week at your section.
- 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.
- Your data will be downloadable via the Labs section of the class webpage. http://online.physics.uiuc.edu/courses/phys552/fall08/
In general, for the Beckman DU experiment, if you did not get “good” results from your data, you should still analyze/present your own data , give reasonable conjectures as to why your data could be off, and explain what you would do differently to test your conjecture. (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 group’s 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. The graduated ND filter calibration is presented at the end.
The optical densities (from which you can calculate concentrations) of the fluorescein solutions are in the table below:
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/
Name OD at 490 nm 1 1. 2 0. 3 0. 4 0.
Lab 1 – Monochromators and such…
I. Introduction
This lab is exploratory and introductory. If you use absorption spectrometers and fluorometers 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 approximate absorption measurements manually, without the automation of modern instruments
Topics Covered
- Monochromators
- Wavelength Dispersion Elements o Diffraction Gratings
- Fluorescein – absorption/fluorescence
References
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)
The Instrument Project: UV-visible spectroscopy. http://www.wooster.edu/chemistry/is/brubaker/uv/default.html
The useful information from a leading monochromator manufacturer http://www.newport.com/Monochromators-and-Spectrographs/370636/1033/catalog.aspx
Eugene Hecht, Optics
II. Monochromator 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 monochromator. Then you will use the monochromator 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
Monochromator Basics
There are two monochromators on the table. Only Monochromator A will be used for Part IIA.
Familiarize yourself with the optical path through the spectrometer. The 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
- light source, 2) entrance/exit slits, 3) mirrors, 4) diffraction grating.
- The schematic should be specific to this monochomator.
- Clearly label all components
- Feel free to be creative, adjusting the light source and focusing lens to get the best result
- 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 k th order maximum at angle ω
given by:
From Ref 1.
2 a sin( ω )= k λ
Note that this is for a special case where the incident angle equals the diffraction angle, the Littrow configuration.
If you want, look for the second order maxima. No extra points will be given for your discovery so you’ll want to do this after you’re done with Lab 1.
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?)
Q2)
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 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 ⋅ 10 −^ OD 0 The optical density is composed of absorbing and scattering components OD = A + S
- The absorption peak of fluorescein is at 490nm.
- 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 A 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 left and right 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 two samples (A and B). 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) What concentration does your measured OD correspond to?
c) 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 given on page 1.
d) 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.
Q4)
cont’d
e) How does the graduated ND filter work? Hint: look at it on its 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 graduated ND filter (i.e., as d changes)?
f) 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, switch to the cuvette marked CONC, 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.).
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 guesstimated 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.
- 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 (see Appendix B for software instructions).
- 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 if you have any questions or problems. 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 (no cuvette), empty cuvette, water, fluorescein cuvette 4. There is a slideable cuvette holder that you can quickly change between samples, but you may need to swap in some cuvettes.
- Use ImageJ to record a mean intensity of the transmitted light for each of the five samples. Save the resulting measurement table.
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.
- Do 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 fluorescein sample 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?
- 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. Make the same measurements for a water solution. 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.
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.
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?
Appendix A – Graduated ND Filter Calibration
d = 0 on the lighter end of the filter. The filter is 22.5 cm long.
OD= 0.47 + 0.09 * d
OD
d (cm)
