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In this lab, you will learn how to use a Perkin Elmer Luminescence LS50B Spectrometer. Use the instrument to determine the concentration of quanine in a given solution, compare absorption to fluorescence spectra, and find out the effect of a quencher on the fluorescence intensity.


Harris, 18.5, 18.6, and J.E. O’Reilly, J. Chem. Ed. 1975, 52, 610.

Apparatus and chemicals

Perkin-Elmer Lambda Luminescence LS50B Spectrometer Fluorescence cuvettes (2) Aluminum foil Kimwipes Labels and marking pen Distilled water H 2 SO 4 solution (1M and 0.05 M) NaCl Quanine or quanine sulfate volumetric flasks and pipettes

an unknown solution of quinine


Fluorescence is the emission of light by a molecule that has absorbed radiant energy; the radiation is emitted at a longer wavelength than the incident absorbed energy. In most fluorescence methods, both the wavelength of the exciting radiation as well as that of the detected fluorescence can be selected. A basic diagram depicting the important photophysical processes is shown in Figure 1.

A ground state singlet molecule, S 0 , absorbs radiation and is excited to accessible

vibrational levels in the excited singlet state, S 1 . Occasionally, excitation to a higher energy level can result in a triplet state, T 1 . Molecules in the excited state (with a lifetime of about 10 -8 s) will drop to the lowest vibrational level of S 1 due to collisions via vibrational deactivation or relaxation. This radiationless transition, labeled R 1 in Figure 1, converts the energy absorbed to heat that is then dissipated through the entire medium.

At the S 1 level, many photophysical processes could occur. Molecules could return to the

highest vibrational level of S 0 that is isoenergetic with the S 1 level via internal conversion (IC). Through another radiationless process, R 2 , the absorbed energy is converted to heat as molecules collide with one another. If the energy states of S 1 overlaps those of T 1 , as illustrated in Figure 1, molecules could cross from the S 1 level to the vibrational level of the T 1 through intersystem crossing (ISC). Following the radiationless vibrational relaxation R 3 , the molecule may finds


itself at the lowest vibrational level of T 1 where it could undergo another intersystem crossing to S 0 . The molecule could then decay back to the lowest vibrational level of S 0 through another radiationless relaxation, R 4 . All of the photophysical processes discussed so far simply convert light to heat.

processes discussed so far simply convert light to heat. Figure 1. Photophysical processes that can occur

Figure 1. Photophysical processes that can occur after a molecule absorbs an ultraviolet or visible radiation. (adapted from Harris, p. 421)

Of interest to us are the photophysical processes that cause light to be emitted, the radiational transition S 1 S 0 called fluorescence and the radiational transition T 1 S 0 called phosphorescence. As to which of the above photophysical processes will dominate depend on the molecule, the solvent and the conditions such as temperature and pressure. The lifetime of fluorescence is always very short (10 8 to 10 4 s) while that phosphorescence could be in the order of 10 4 to 100 s. Fluorescence or phosphoresce can be quenched (decrease in intensity) by the presence of some ions or molecules. Quenching is another radiationless deactivation process involving interaction with some sort of quencher.

The quantum yield of fluorescence, is given by













where k F is the rate of fluorescence emission, k ISC is the rate of intersystem crossing , k IC is the rate of internal conversion and k Q is the rate of quenching. The influence of the k Q increases with the concentration of the quencher [Q]. Molecules that have small k ISC will generally be strongly fluorescent. Saturated molecules and molecules with only one double bond do not exhibit



significant fluorescence. Molecules with at least one aromatic ring or multiple conjugated double bonds are prone to have fluorescence spectra in the visible or near ultraviolet regions. Phosphorescence often is associated with solid materials (including those solidified by freezing).

Fluorescence spectroscopy is very sensitive and is generally more sensitive than techniques that measure absorption. The amount of detected fluorescence, F, is related to the amount of radiation absorbed and the fluorescence quantum yield,


K P P K P 1 10






where K is a constant dependent on instrument parameters. For dilute solutions ( bc 0.01), the equation reduces to


KP 2.303 bc



The fluorescence intensity is then linearly related to the concentration of the absorbing species and the intensity of incident radiation, P 0 . A plot of F vs. concentration is used as a calibration curve for quantitative analyses.

In modern fluorescence instruments, primary monochromator is placed in front of the excitation lamp source, usually a xenon lamp, to select the wavelength of excitation light. In order to separate the emitted radiation from the incident light, fluorescence measurements are made at right angles to the incident beam. This is possible because fluorescence is emitted in all directions, but the incident radiation passes straight through the cell. In addition a second monochromator is usually located before the fluorescence detector, allowing the emitted radiation to be wavelength resolved. In an excitation spectrum, the secondary monochromator selects a fixed detection wavelength while the primary monochromator is scanned. The result is analogous to an absorption spectrum but it is detected indirectly via fluorescence rather than directly and so may be complicated by nonradiative processes. In an emission spectrum, the excitation wavelength is held fixed while the secondary monochromator is scanned and the fluorescence is wavelength resolved.

In the lab

1. Prepare 100.0 g/mL stock solution of quinine. How many grams of quinine sulfate dihydrate or of quinine (depending on which is available) is needed to prepare a 250-mL solution? Weigh out the appropriate amount, add 12.5 mL of 1 M H 2 SO 4 and dilute to mark with water. Quinine solutions must be prepared fresh daily and should be protected from light. All containers must be covered by Al foil.


2. Prepare a series of quinine standard solution (each with a total volume of 10 mL) from the 100.0 g/mL stock. Make a sequential tenfold dilution with 0.05 M H 2 SO 4 , i.e. use the 100 g/mL solution to prepare the 10 g/mL, the 10 g/mL to prepare the 1 g/mL and so on. Prepare solutions with concentrations of 10, 1, 0.1, 0.01, 0.001, and 0.0001 g/mL. Obtain the fluorescence emission spectrum for each of the above dilutions, using 0.05 M H 2 SO 4 as a blank. Set the wavelength of excitation to 250 or 350 nm (you may choose) and scan from 260 to 600 nm or 360 to 600 nm, respectively. Be sure to save each spectrum both in the default binary format and in ASCII format so you can take them into Excel later. What is the peak of the emission spectrum, the emission max ?



3. Obtain a quinine solution of unknown concentration and measure its fluorescence emission spectrum making proper dilutions if necessary. Utilize the same settings used in step 2. Again, save the spectrum in binary and ASCII formats.

4. Measure the fluorescence excitation spectra of the 1 g/mL quinine solution; let the primary monochromator scan from 200 to 440 nm with the secondary monochromator set at 450 nm. What is/are the peak(s) in the fluorescence excitation spectra, the excitation max ’s? Next obtain its UV-vis absorption spectra. Set the UV-vis spectrometer to run from 700 nm down to 200 nm, with an increment of 5 nm. Do not forget to AUTOZERO the instrument. What is/are the peak(s) in the absorption spectra, the absorption max ’s? Save both the absorption and fluorescence excitation spectra in binary and ASCII formats.

5. Prepare six solutions (5 mL total volume) all containing 1 g/mL quinine and each containing 0, 100, 300, 1000 and 2000 NaCl g/mL. Tell me how you would do this given that you have a 5 M NaCl stock solution, you must show the necessary calculations. Obtain the fluorescence emission spectrum for each of the six solutions, using 0.05 M H 2 SO 4 as blank. Should our blank contain NaCl as well? Why did we not include NaCl in the blank? Employ the same settings used in part 2. Once again, save each spectrum in binary and ASCII formats.

WASTE DISPOSAL: Solutions must go to a “quinine” waste bottle.

Data Analysis

Using the ASCII files, load all your spectra into a single Excel workbook. The easiest thing will be to use one workbook with sheets of data. Each sheets containing the data for each part of the experiment.

A. Varying Concentrations.

1. Create a chart that shows all the spectra varying the concentration (your Figure 1). Give at least two reasons why we used very dilute solutions. The Fluorescence intensity often varies with the instrument used and the conditions of the experiment so often relative fluorescence intensities are reported. To do this let the most concentrated solution have a 100 % relative fluorescence. With this as basis, present all spectra in terms of % relative fluorescence intensities. Use a scatter chart type, with the points connected by lines but no markers. Make sure you have a legend that enables both of us to know which line corresponds to which spectrum. Estimate the error in the relative intensity. What is the signal-to-noise ratio (S/N)?

2. Make a new chart. On this chart, plot the relative intensity at the emission max for each spectrum against the concentration (your Figure 2). Fit the data to a straight line, and report the value of the slope and intercept, along with their standard errors. Do you expect this plot of relative fluorescence intensity vs. concentration to be linear? What, if any, could cause a deviation from linearity? Justify your answer (whenever necessary, always justify your answers).

3. Within the precision of the wavelength measurements in your spectra, did the emission max vary with concentration? What is your estimate of the error in the wavelength?



4. Repeat steps 1 and 2, except instead of using peak height as the measure of the fluorescence intensity use the area under the curve (remember how you did it in your Excel exercise). This means youKll have to integrate the each of the fluorescence spectra. Plot the relative intensity, in terms of peak area, for each spectrum against the concentration (your Figure 3). Did you notice any discrepancies between the two methods of estimating fluorescence intensity? Which do you think is the better method? Explain your answer.

5. Overlay the spectrum of your unknown onto Figure 1. Show clearly how you calculated the concentration of your unknown, based on Figures 2 and 3, respectively. Do not forget to include the uncertainty.

B. The Absorption, Emission and Excitation Spectra.

6. Now create a new chart that shows the absorption, fluorescence emission and fluorescence excitation spectra superimposed (your Figure 4). The y-axis for each of these spectra is not the same. Multiply the absorption spectra with a factor, say 10 (the choice is yours), so that all the peaks will be visible (and not show as a flat line). Comment on the differences and similarities.

C. Effect of Adding Quenchers.

7. Create a chart that shows the intensity of quinine fluorescence emission as a function of NaCl concentration (your Figure 5). Again, make sure you have a legend that enables both of us to know which line corresponds to which spectrum. Within the precision of the wavelength measurements in your spectra, are the peaks all at the same wavelength? Did you expect this result? Account for any discrepancies.

8. Make a new chart. On this chart, plot the relative intensity at the emission max for each spectrum against the NaCl concentration (your Figure 6). Fit the data to a straight line, and report the value of the slope and intercept, along with their standard errors. Did you expect this plot of relative fluorescence intensity vs. NaCl concentration to be linear? What happened to the fluorescence intensity of the quinine solution as the amount of NaCl increased? What would be the consequence if the unknown you used had quinine dihydrochloride instead of quinine sulfate?

Your report will include:

a brief introduction;

a summary of the experimental section, describing deviations from the given procedure, the

schematic of the instrument and supplementary information; six figures in the results section and some tables (depending on how you would like to your

data presented); show sample calculations but do not include spreadsheets here. the discussion, do not forget to answers all the questions; include a discussion of possible

sources of error and background correction. any references you have footnoted;

In addition to your written report, turn in your spreadsheet(s) and the entire spectrum files (in both binary and ASCII format). Do not print out your workbook.