FLUORESCENCE SPECTROSCOPY

Aim

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.

Readings

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
H2SO4 solution (1M and 0.05 M)
NaCl
Quanine or quanine sulfate
volumetric flasks and pipettes
an unknown solution of quinine
Theory
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, S0, absorbs radiation and is excited to accessible
vibrational levels in the excited singlet state, S1. Occasionally, excitation to a higher energy level
can result in a triplet state, T1. Molecules in the excited state (with a lifetime of about 10-8 s) will
drop to the lowest vibrational level of S1 due to collisions via vibrational deactivation or
relaxation. This radiationless transition, labeled R1 in Figure 1, converts the energy absorbed to
heat that is then dissipated through the entire medium.
At the S1 level, many photophysical processes could occur. Molecules could return to the
highest vibrational level of S0 that is isoenergetic with the S1 level via internal conversion (IC).
Through another radiationless process, R2, the absorbed energy is converted to heat as molecules
collide with one another. If the energy states of S1overlaps those of T1, as illustrated in Figure 1,
molecules could cross from the S1 level to the vibrational level of the T1 through intersystem
crossing (ISC). Following the radiationless vibrational relaxation R3, the molecule may finds
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itself at the lowest vibrational level of T1where it could undergo another intersystem crossing to
S0. The molecule could then decay back to the lowest vibrational level of S0 through another
radiationless relaxation, R4. All of the photophysical processes discussed so far simply convert
light to heat.

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 S1 o S0 called fluorescence and the radiational transition T1 o S0
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
kF
)F
k F  k ISC  k IC  k Q [Q]
where kF is the rate of fluorescence emission, kISC is the rate of intersystem crossing , kIC is the
rate of internal conversion and kQ is the rate of quenching. The influence of the kQ increases with
the concentration of the quencher [Q]. Molecules that have small kISC will generally be strongly
fluorescent. Saturated molecules and molecules with only one double bond do not exhibit
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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,
F 
K) F P0  P  K) F P0 1  10 Hbc 
where K is a constant dependent on instrument parameters. For dilute solutions (Hbc d 0.01), the
equation reduces to
F KP0 2.303Hbc) F
The fluorescence intensity is then linearly related to the concentration of the absorbing species
and the intensity of incident radiation, P0. 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 Pg/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 H2SO4 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.
WARNING! QUININE IS HARMFUL IF SWALLOWED. MAY BE HARMFUL IF
INHALED. AFFECTS CARDIOVASCULAR AND CENTRAL NERVOUS SYSTEM.
MAY CAUSE IRRITATION TO SKIN, EYES, AND RESPIRATORY TRACT.
2. Prepare a series of quinine standard solution (each with a total volume of 10 mL) from
the 100.0 Pg/mL stock. Make a sequential tenfold dilution with 0.05 M H2SO4, i.e. use
the 100 Pg/mL solution to prepare the 10 Pg/mL, the 10 Pg/mL to prepare the 1 Pg/mL
and so on. Prepare solutions with concentrations of 10, 1, 0.1, 0.01, 0.001, and 0.0001
Pg/mL. Obtain the fluorescence emission spectrum for each of the above dilutions, using
0.05 M H2SO4 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 Omax?
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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 Pg/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
Omax’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 Omax’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 Pg/mL quinine and each
containing 0, 100, 300, 1000 and 2000 NaCl Pg/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 H2SO4 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 Omax 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
Omax vary with concentration? What is your estimate of the error in the wavelength?

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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 you’’ll 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 Omax 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:

x a brief introduction;
x a summary of the experimental section, describing deviations from the given procedure, the
schematic of the instrument and supplementary information;
x 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.
x the discussion, do not forget to answers all the questions; include a discussion of possible
sources of error and background correction.
x 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.

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