CHEM 311L

Revision 2.0

A Fluorometric Analysis of Quinine in Tonic Water

In this Laboratory Exercise, we will determine the amount of Quinine in Tonic Water using a
fluorometric analysis. Fluorescence Spectroscopy is an extremely sensitive technique and
Quinine is one of the most active fluorescers known. Thus, the determination of even small
amounts of Quinine is relatively easy. As is typical of Spectrophotometric analyses, we will first
prepare a calibration curve for the Fluorescence Intensity of Quinine and then use this curve to
determine the Quinine concentration in a diluted sample of Tonic Water. Finally, a urine sample
taken within 24 hours of a subject's ingestion of tonic water will be tested for its Quinine content.
This can be compared with the amount of Quinine injested.

Luminescence involves emission of photons from excited atoms or molecules. Fluorescence and
Phosphorescence, both luminescent processes, involve emission of photons from systems that
have been excited by absorption of photons. In molecular Fluorescence Spectroscopy, the
analyte molecule first absorbs a photon (excitation, Eexcite) that leaves the analyte in an
electronically and vibrationally excited state.
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At this point, the molecule rapidly looses excess vibrational energy by non-radiatively relaxing
to the ground vibrational level of the excited electronic state. This occurs because energy is
transfered to solvent molecules as the analyte jostles against them. This relaxation process is
very efficient and very rapid. Finally, the molecule can fluoresce (Erelax). Alternatively, the
molecule can undergo a non-radiative transition (Internal Conversion) to the ground state.
Molecules undergoing Internal Conversion transit to the Ground State without emitting radiation.
This is an efficient relaxation process when higher vibrational states of the Ground Electronic
State overlap with lower vibrational states of the Excited Electronic State.

Because of the non-radiative relaxation in the electronically excited state, the excitation energy is
always larger than the relaxation energy.

Eexcite > Erelax (Eq. 1)

Because the energies of the photons involved in these transitions are related to their wavelengths
via:

 Ephoton = h c /  (Eq. 2)

(c is the speed of light and h is Planck’s constant), the wavelength of an exciting photon is
always shorter than that of a photon emitted during relaxation:

excite < relax (Eq. 3)

Quinine is an example of a molecule that undergoes both fluorescence and internal conversion.
It has two possible excitation wavelengths; 250 and 350 nm. However, because internal
conversion is very efficient between the two electronically excited states, only a single emission
at 450 nm (blue) is observed.
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Tonic Water Containing Quinine Fluorescence of Quinine Excited by UV Radiation

(http://en.wikipedia.org/wiki/File:Tonic_water_uv.jpg)

Fluorescence spectra can be measured using a Spectrofluorometer. Light from the source is
dispersed and the excitation wavelength is selected using a monochrometer. The excitation
radiation impinges upon the sample, which then begins to fluoresce. Fluorescent radiation is
itself dispersed and the spectrum is measured using an appropriate detector.
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In an actual spectrofluorometer, the dispersing element is usually a diffraction grating. In

simpler Fluorometers, the dispersing elements are replaced with filters.

The Flourescent Intensity (F) will be proportional to the radiant Power absorbed by the sample
(Po-P):

F = K’ (Po – P) (Eq. 4)

Inserting Beer’s Law:

P/Po = 10-bc (Eq. 5)

and expanding the exponential term, gives us:

F = K’ Po {2.3bc – (2.3bc)2/2 - ...} (Eq. 6)

Provided the sample Absorbance is relatively low, we can truncate the expansion:

F = 2.3 K’ Po bc (Eq. 7)

When Po is constant, we see the Fluorescent Intensity is proportional to the Concentration of the
analyte:

F = Kc (Eq. 8)

This, then, provides a method for quantifying the amount of analyte in a system based on
fluorescence measurements.

A few words of caution. If the concentration of the analyte is high enough, higher order
expansion terms become important and the relationship between F and c is no longer linear.
And, if the concentration becomes very high, the system begins to absorb its own emitted
radiation, causing a decrease in fluorescence intensity and severe non-linearities set in.

Fluorescence spectroscopy is much more sensitive than corresponding Absorbance spectroscopic

techniques. This is because light emitted against a dark background (fluorescence) is much
easier to detect than a slight dimming of intensity against a light background (absorbance).
Imagine the difference between observing a photographers flash going off in a dark sports
stadium (fluorescence) versus trying to detect the same flash on a bright sunny day (absorbance).
However, fluorescence techniques are severely limited by the number of analytes that actually
fluoresce. Most systems shed their excitation energy via radiationless pathways. Structurally,
molecules that possess unsubstituted aromatic rings or other structurally rigid elements have a
propensity for fluorescing. Fused-ring heterocycles also fluoresce nicely.
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Finally, other species in solution can act as quenching agents (Q); they absorb radiation non-
radiatively from the excited analyte (S*) causing a decrease in the fluorescence intensity.

S + hexcite S* (excitation)

S* S + hrelax (fluorescence)

or

S* + Q S+Q (quenching)

It can be shown the ratio of unquneched-to-quenched fluorescent intensities (Fo/F) is related to

the quenching agent concentration ([Q]) via (Stern-Volmer Relation):

(Fo/F - 1) ~ [Q]

The proportionality is related to the fluorescence lifetime and the rate constant for the quenching
process.

In many cases, Oxygen (O2) acts as a very effective quenching agent for those systems that do
fluoresce. Fortunately for our experiment, O2 does not quench Quinine fluorescence. But,
Chloride Ion (Cl-) does act as a quenching agent for Quinine. Thus, it is imperative to keep the
Chloride Ion concentration low throughout. In particular, we should not use the Quinine
Hydrochloride, a common salt of Quinine, to prepare our standard solutions.

Quinine, as already mentioned, is a very efficient fluorescer with excitation wavelengths at 250
and 350nm. Emission occurs at 450nm. Quinine, originally
derived from the bark of the Cinchona tree, is an effective
cure for malaria. Traditionally, because of its bitter taste,
Quinine was mixed with gin, giving rise to the Gin and
Tonic cocktail. Quinine is used as a flavoring agent in
Tonic Water, Bitter Lemon and Vermouth. This is limited
by the Food and Drug Administration to 83ppm; with most
commercial preparations at 25 – 60ppm. We will measure
the amount present in a commercial product.

Quinine
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As usual, we will first prepare a calibration curve for the Fluorescence Intensity at various
Quinine concentrations, all prepared from a stock solution of Quinine Sulfate Dihydrate at 100
ppm. Then, our Tonic Water will be diluted so as to produce a Fluorescence Intensity lying on
the calibration curve. This final measurement will be used to determine the concentration of the
Quinine in the Tonic Water.

Additionally, the urine of a human subject who has consumed a small bottle of Tonic Water will
be tested for excreted Quinine using the same calibration curve. As noted by James E. O'Reily,
"Screening of urine for quinine is a fairly common method for effective surveillance of heroin
abuse within a narcotic control, treatment, or aftercare program, since quinine is a common
diluent of illegal heroin samples." (J. Chem. Ed. 52, 1975, 611) It has been noted by several
sources that Quinine can be detected in urine by flourospectroscopy for several days following
consumption of this amount of Tonic Water.

All fluorescence measurements will be made using the Photon Technology International system
pictured below.
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Pre-Lab Calculations

The Mineral Museum on campus has several very nice examples of fluorescent minerals.
They are located in the "dark room" in the back corner of the museum. You should stop
by and observe the minerals while fluorescing. In particular, note the excitation
wavelengths and the color (wavelength) of the fluorescence. Also note that a few of the
minerals phosphoresce. Note that the key difference between fluorescence and
phosphorescence is the much longer lifetime of the phosphorescence process.

1. Nominally, a plot of Fluor. Intensity vs Conc. should be linear, at low analyte

concentrations. However, we are preparing a graph of log(Fluor. Intensity) vs
log(Conc.) for our calibration curve. Why are we doing this and what effect will
this have on the linearity of the plot?

2. What is the total dilution of the Tonic Water used in our procedure?
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Procedure

Preparation of Quinine Standards

1. A 100.0 ppm stock solution will be prepared, from which several standard dilutions will be
made. The stock solution is prepared by accurately weighing 120.7 mg of Quinine Sulfate
Dihydrate, tansfering to a 1L volumetric flask, adding 50 mL of 1M H2SO4 and diluting to
the mark with Distilled Water. This stock solution should be prepared daily and be
protected from the light.

2. A series of 10X dilutions of the Quinine stock should be prepared with 0.05M H2SO4.
After each dilution, measure the Fluorescence. Continue with the dilutions until the
Fluorescence intensity is approximately that of the blank.

3. Prepare a graph of log(Rel. Fluor. Intensity) vs log(Concentration). This is your calibration

curve. Discuss any deviations from linearity with your instructor. Do a Linear Least
Squares Analysis of the linear portion of the curve only.

Preparation of the Tonic Water

1. Pipet 5.00 mL of Tonic Water into a 250 mL Volumetric flask. Dilute to the mark with
0.05M H2SO4 and mix thoroughly.

2. Pipet 5.00 mL of the above solution into a 25 mL Volumetric flask. Dilute to the mark
with 0.05M H2SO4 and mix thoroughly.

3. Record the Fluorescence intensity of this solution.

4. Determine the Quinine concentration using your prepared calibration curve.

Collection of Fluorescence Data

1. Your laboratory instructor will demonstrate the use of the Fluorometer. Quinine has two
excitation wavelengths; 250 and 350nm. Either can be used. The wavelength of maximum
fluorescence for Quinine is 450nm.

Preparation of the Urine Samples

The following procedure is adapted from "Fluorescence Experiments with Quinine" by James E. O'Reily; J. Chem.
Ed. 52 (1975) 610.

1. A small bottle of Tonic Water (8 oz) should be consumed approximately 24 hours prior to
the laboratory session.
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2. Obtain a urine sample cup and an alcohol wipe from your laboratory instructor and proceed
to the restroom to obtain a urine sample of at least 5 mL. Be sure to completely swab the
outside of the sample cup with the alcohol wipe after obtaining this sample.

3. Working over an absorbent lab bench cover, pipet 2 mL of urine into a 15 mL centrifuge
tube and adjust the pH to 9-10 with 3.7 M NH4OH.

4. Add 4 mL of a 3:1 chloroform-isopropanol mix and shake the sample for ~1 minute.
Allow the layers to separate. Centrifuge if needed.

5. Transfer 2 mL of the lower organic phase to a clean, dry centrifuge tube and add 2 mL of
0.05 M H2SO4. Shake the tube for ~1 minute and then allow the layers to separate.

6. The upper aqueous layer is transferred to a cuvette and its fluorescence emission is
measured.

7. Perform the same procedure on 2 mL of Distilled Water and determine the fluorescence
emission of this blank sample.

8. Dispose of all samples and treat all glassware as directed by your laboratory instructor.

9. O'Reilly reports there is a fluorescence interferent which emits at 425 nm, overlapping with
our 450 nm signal. He further reports that this amounts to ~0.4 ppm of Quinine in the
original urine sample. It is also reported this method of extracting Quinine from urine is
about 95% effective.

Use your prepared calibration curve to determine the amount of Quinine in your urine
sample. Correct for both of the above affects. Report the amount of Quinine ingested, the
time after Quinine ingestion the urine sample was taken and the Quinine level in the urine
in ppm.