Fluorometric Assay of Quinine

Purpose: Instruments capable oI measuring Iluorescence are known as Iluorometers or

Iluorescence spectrometers. They all have similar construction. The basic components
are: (1) a radiant energy source (usually Xe or Hg arc lamp), (2) a device to isolate the
Excitation Wavelength(s) oI light, (3) a sample holder, (4) a device to isolate the
Emission Wavelength(s) oI light, and (5) a photodetector to measure the Iluorescence.
Consult your textbook Ior general instrument schematics and Iurther inIormation.
"True" Iluorescence spectra are obtained when a correction Ior the lamp output vs.
wavelength is made. The spectroIluorometer used here provides this correction
automatically. At very low concentrations oI Iluorophore, the power oI the emitted
light is directly proportional to the amount or concentration oI Iluorophore. However,
a variety oI conditions adversely aIIect this linear relationship;
O selI-quenching
O external quenching
O decreased quantum eIIiciency
O contaminants.
As always, clean glassware and spectral purity oI reagents are requirements Ior
accurate Iluorometry.
The alkaloid base quinine (or quinidine) can be extracted Irom an alkaline solution oI
a biological matrix using the dichloromethane-isopropanol solvent system. A second
extraction using this solvent system with dilute acid (0.1 N H
2
SO
4
) transIers virtually
all the alkaloid into the acid layer. The emission Iluorescence spectrum oI this acid
solution can then be used to identiIy and quantitate the extracted compound.
#eferences: R. F. Chen, Analytical Biochemistry 19, 374-387 (1967), I. Sunshine,
Ed., Methodology for Analytical Toxicology, Chemical Rubber Co. (1976),
Application Data Sheet, "Clinical Analysis Ior Quinidine in Blood," M8090, Beckman
Instruments, Fullerton, CA.
Equipment: Varian model SF-330 spectroIluorometer, centriIuge, plastic centriIuge
tubes
Samples: 10 mL quinine water, 10 mL urine

#eagents
O Quinine SulIate, Stock Standard: 100 mg/L. Weight out 30.2 mg quinine
sulIate
.
2H
2
O and dissolve in 250 mL 0.1 N H
2
SO
4
.
O Quinine Standards, Working: 5, 10 and 15 mg/L. Using 100 mL volumetric
Ilask, dilute appropriate volumes oI the quinine stock standard using 0.1 N
H
2
SO
4
as diluent. Mix thoroughly by inversion.
O SulIuric Acid: 0.1 N. Add 9.0 mL dilute H
2
SO
4
(6 N) to 500 mL volumetric
Ilask and dilute to the mark with distilled water. Mix.
O Extraction Solvent: Mix 3 volumes oI dichloromethane with 1 volume oI
isopropanol. Mix thoroughly. Do not make any more than you need. (4 mL
solvent/test.)
O pH 8.5, 0.1 M Ammonia BuIIer: Dissolve 5.4 g NH
4
Cl in 90 mL (use a 100 mL
vol. Ilask) distilled water, then add 2.5 mL 6 N NH
4
OH. Adjust to pH 8.5 using
6 NNH
4
OH or 6 N HCl. Dilute to mark with distilled water and mix.
O Quinine Control: bottled quinine water (Canada Dry).
O Urine Unknown: Further explanation oI this sample will be provided at the
beginning oI the laboratory exercise.

Procedure
1. Label a series oI 16 x 125 mm screw-cap test tubes as: Blank, STD-5, STD-
10, STD-15, Urine, Control.
2. Add 2.0 mL oI appropriate sample to each tube, using distilled water Ior the
BLANK. Then add 1.0 mL ammonia buIIer to each oI the six tubes and mix.
3. Add 4.0 mL oI extraction solvent to all tubes, caps, and mix by inversion Ior 5-
10 minutes.
4. CentriIuge the tubes Ior 2 minutes at 500 RPM using a clinical centriIuge. Use
a setting oI 8 on "Speed Control."
5. Aspirate the (UPPER) aqueous layers and then decant each (LOWER) organic
layer into a clean, correspondingly labeled 16 x 125 mm screw-cap tubes.
6. Add 4.0 mL oI 0.1 N H
2
SO
4
to each tube. Cap tubes, and mix by inversion Ior
5-10 minutes.
7. CentriIuge tubes Ior 2 minutes and 1500 RPM.
8. TransIer each (UPPER) aqueous layer to a labeled test tube by means oI a
Pasteur pipet.
9. Start the spectroIluorometer, Iollowing the instructions as listed in the
instruction manual. Consult the instruction manual Ior guidelines on setting slit
widths and scan speeds.
10.Measure the spectra as listed below;
O Emission spectra oI quinine in 0.1 N H
2
SO
4
: Use 10 mg/L standard as
originally prepared. Set the excitation wavelength to 350 nm and scan Irom 200
nm to 750 nm using the emission monochromator.
O Excitation Spectra oI quinine in 0.1 N H
2
SO
4
: Using the same solution, and an
emission wavelength near the maximum Iound Irom the above step, scan the
excitation wavelength Irom 200-340 nm.
O Set the excitation wavelength to 350 nm and perIorm an emission scan oI blank
solution Irom 300-700 nm.
O Set the excitation wavelength to 350 nm. Set emission wavelength to the
optimum wavelength Iound Irom the emission scan. Place 15 mg/L extracted
standard in the cuvette and adjust controls to just keep recorder pen on scale.
Now run an emission scan Irom 300-600 nm.
O With controls adjusted, run emission scans on all remaining solutions including
control and unknown.

alculations
O Measure the Iluorescence signal oI all solutions and prepare table as shown
below:
Solution
Solution
Fluorescence
Total
Fluorescence
Corrected Quinine
Conc. (mg/L)
Blank
STD-5
STD-10
STD-15
Control
Unknown
O Plot corrected Iluorescence signal as a Iunction oI concentration (mg/L) and
determine concentration oI control and unknown in mg/L.
O IdentiIy; a) Raleigh scattering peak, b) Raman spectra, and c) second order
spectra, in the blank emission spectrum.
O Comment on spectra oI control and urine relative to pure standard spectra.

omments
The silica cuvettes used in this experiment are expensive. Please treat them with
utmost care.
To determine the correct excitation wavelength is not a trivial process. However, a
judicious selection initially may save one large amounts oI time. One starting point is
that oI the ultraviolet absorption spectrum Ior the compound in question. The
excitation wavelength values are logical selections. Later, these wavelengths can be
veriIied in the excitation wavelength scan.
The selection oI both excitation and emission wavelengths involves determining the
sensitivity, onset oI quenching, interIerence, etc. It is appropriate in methodology set-
up to scan the solution thoroughly beIore deciding on Iixed wavelengths Ior
measurement.

#eport
Absorption, emission, Iluorescence, phosphorescence, luminescence,
photoluminescence, chemiluminescence, thermal relaxation, intersystem crossing, and
internal conversion are commonly used terms in spectroscopy. DeIine each term. How
are the terms related? Explain the chemistry involved in each process. What are the
time scales involved with each process?
A discussion oI the kinetics oI Iluorescence should be included in all reports. In your
conclusions, comment on the utility and sensitivity oI Iluorescence. Those oI you who
wish to do your Iormal report on Iluorescence spectroscopy should consider the
Iollowing ideas.
1. Compare the absorbance spectrum with the Iluorescence spectrum. What are
the similarities and diIIerences? Account Ior these phenomena.
2. A discussion on Iluorescence quenching could be given.
3. Why do chemicals Iluoresce? Do all chemical species Iluoresce?
4. What is a Jablonski diagram?