Fluorometric Determination of Quinine
The purpose of this experiment is to determine the concentration of quinine in tonic water using
the method of spectrofluorometry.
Preparation of Stock Solutions
1. Prepare ~100 mL of ~1 M sulfuric acid* and mix well.
2. Prepare ~1 L of ~0.05 M sulfuric acid and mix well.
3. Prepare a 100.0 µg/mL quinine stock solution by quantitatively transferring exactly
0.1180 g of quinine sulfate [(C20H24N2O2)2•H2SO4•2H2O, FW 782.94] to a 1-L
volumetric flask, adding ~50 mL of your ~1 M sulfuric acid, and diluting to volume with
deionized water. Mix the solution thoroughly. (Be sure to write down the complete
formula for this compound, which will be needed for your report. )
Preparation of Quinine Standard Solutions
1. Prepare five solutions using successive 10-fold dilutions of your quinine stock solution
and appropriate volumetric glassware (5 mL pipet, 50 mL volumetric flasks). Dilute each
to volume using your ~0.05 M sulfuric acid. Mix each solution thoroughly before using
it to make the next solution. Quinine concentrations in your resultant five solutions
should range from 1.000 x 101 µg/mL through 1.000 x 10–3 µg/mL.
Preparation of Tonic Water Solutions
1. Pipet 1.00 mL of flat tonic water into a 100-mL volumetric flask and dilute to volume
using your ~0.05 M sulfuric acid. Mix well.
2. Prepare two solutions using successive 5-fold dilutions of your tonic water solution
(prepared above) and appropriate volumetric glassware (10-mL pipet, 50 mL volumetric
flasks). Dilute each to volume using your ~0.05 M sulfuric acid. Mix the first solution
thoroughly before using it to make the second solution.
* Concentrated sulfuric acid contains 95-98% H2SO4 by weight and has a density of 1.840 g/mL. H2SO4 has a
formula weight of 98.08 g/mol.
Instrument Operation, Varian Cary Eclipse Fluorometer
1. Turn on the power switch of the Cary Eclipse Fluorometer.
2. Click the window start button, point to program, and select Cary Eclipse.
3. Click on Scan shortcut.
4. Select the setup button to displace the setup dialog and specify the method parameters.
Set Data Mode to Fluorescence. Set the scan setup mode to emission. Set the X Mode
to wavelength (nm). Enter the Excitation (nm) value of 350 nm. Enter an Excitation
slit (nm) value of 5 and an Emission slit (nm) value of 5. Enter a start (nm) value of
360 nm and enter a stop (nm) value of 520 nm. Clear the 3-D mode check box. Select
the Scan Control to Medium.
5. Select the options tab to display the options page. Select the overlay traces check box
to overlay the results of the traces on one graph. Then clear CAT or S/N Mode, Cyclo
Mode and Smoothing check boxes.
6. Set the Excitation filter to Auto. Set the Emission filter to Open. Set the PMT
Detector Voltage to Medium.
7. Select OK to close the setup dialog.
1. Place the blank solution in the sample compartment #4.
2. Click the zero button to zero the system. When the result is zeroed, the work ‘Zeroed’
will appear in the Y display box in the top left corner of the scan application window. DO
NOT discard the blank, you must zero the system before each reading.
3. Place your most dilute sample in the sample compartment.
4. Select the start button to commence a data collection. When the sample name dialog is
displayed, enter an appropriate name for your sample and select OK. The scan will
commence and the trace will appear in the graphics area.
5. By tracing along the scan with the cursor, record the intensity (a.u.) at 450 nm.
6. Repeat steps 1-5 for all five quinine standard solutions and the two tonic water solutions,
measuring in order from the most dilute to the most concentrated solutions. Be sure to
rinse the sample cuvette several times with each new solution to avoid contamination.
1. Remove and clean the cuvettes and return them to the instructor.
2. Exit the program, and shut down the computer, temperature control, and instrument.
Data Treatment/Report includes the following:
1. All stock solution preparation calculations, including a calculation for the concentration
of concentrated sulfuric acid using the footnote data on the previous page. Don’t forget
to use the appropriate number of significant figures for the glassware used, whether
volumetric (e.g., pipet or volumetric flask) or not (e.g., graduated cylinder).†
2. A calculation and explanation demonstrating why a mass of 0.1180 g of quinine sulfate is
needed to prepare a quinine stock solution with a concentration of 100.0 µg/mL. Note:
just one of the two waters of hydration escapes into solution when the quinine sulfate
3. A table listing Quinine Standard concentrations and measured Fluorescence Intensities
(a.u.). Include a sample calculation showing how your performed a 10-fold dilution.
4. A table listing number of 5-fold Tonic Water dilutions and measured Fluorescence
Intensities (a.u.). Include a sample calculation or description showing how your
performed a 5-fold dilution.
5. A calibration curve for quinine: a log-log plot of Fluorescence Intensity (a.u.) vs.
Quinine Concentration (µg/mL) for your five quinine standard solutions. Note: some of
these solution concentrations are purposely too high or low to register properly,
according to the detection limits of the instrument. If too high, they are off scale; if too
low, they are lost in the noise of the instrument or even give a negative intensity. Plot
data from only those solutions which fall within the normal detection range of the
instrument, i.e., which show a Fluorescence Intensity that changes in a reasonable way as
Quinine Concentration changes. Important: plot your data as is (i.e., using actual
quinine concentrations and measured absorbance intensity values), then use the
Excel scale feature to change the x and y scales to a log scale.
6. Although the plot above is not linear overall, a section of it will appear linear to the eye
on your log-log plot. Construct a second plot using just those data that appear linear (if
different from your calibration curve), and determine the equation of the line using the
Trendline feature of Excel. Show the equation on your plot. Make sure you have at least
four sig figs each in your slope and intercept of your line to use in your calculations
7. Use the equation of your line and the measured absorbance intensities of your tonic water
solutions, determine the quinine concentration in both tonic water solutions.
8. Properly accounting for dilution, calculate the concentration of quinine (in µg/mL) in the
original sample of tonic water in both solutions. While these should ideally be
identically, they probably will not be. Make a quantitative comparison of your two
results, i.e., report the percent error between them (difference/average x 100%).
† See chapter of Harris (pages 25 and 27) for tolerances of volumetric glassware.