Fluorometric Assay of Quinine
Purpose: Instruments capable of
measuring fluorescence are known as fluorometers or fluorescence
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) of
light, (3) a sample holder, (4) a device to isolate the Emission
Wavelength(s) of light, and (5) a photodetector to measure the
fluorescence. Consult your textbook for general instrument
schematics and further information.
"True" fluorescence spectra are
obtained when a correction for the lamp output vs. wavelength is
made. The spectrofluorometer used here provides this correction
automatically. At very low concentrations of fluorophore, the
power of the emitted light is directly proportional to the amount
or concentration of fluorophore. However, a variety of conditions
adversely affect this linear relationship;
- external quenching
- decreased quantum efficiency
As always, clean glassware and spectral purity
of reagents are requirements for accurate fluorometry.
The alkaloid base quinine (or quinidine) can be
extracted from an alkaline solution of a biological matrix using
the dichloromethane-isopropanol solvent system. A second
extraction using this solvent system with dilute acid (0.1 N
H2SO4) transfers virtually all the alkaloid
into the acid layer. The emission fluorescence spectrum of this
acid solution can then be used to identify and quantitate the
References: 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 for Quinidine in
Blood," M8090, Beckman Instruments, Fullerton, CA.
Equipment: Varian model SF-330
spectrofluorometer, centrifuge, plastic centrifuge tubes
Samples: 10 mL quinine water, 10 mL urine
- Quinine Sulfate, Stock Standard: 100 mg/L.
Weight out 30.2 mg quinine sulfate . 2H2O
and dissolve in 250 mL 0.1 N H2SO4.
- Quinine Standards, Working: 5, 10 and 15
mg/L. Using 100 mL volumetric flask, dilute appropriate
volumes of the quinine stock standard using 0.1 N H2SO4
as diluent. Mix thoroughly by inversion.
- Sulfuric Acid: 0.1 N. Add 9.0 mL
dilute H2SO4 (6 N) to 500 mL
volumetric flask and dilute to the mark with distilled
- Extraction Solvent: Mix 3 volumes of
dichloromethane with 1 volume of isopropanol. Mix
thoroughly. Do not make any more than you need. (4 mL
- pH 8.5, 0.1 M Ammonia Buffer:
Dissolve 5.4 g NH4Cl in 90 mL (use a 100 mL
vol. flask) distilled water, then add 2.5 mL 6 N
NH4OH. Adjust to pH 8.5 using 6 N NH4OH
or 6 N HCl. Dilute to mark with distilled water
- Quinine Control: bottled quinine water
- Urine Unknown: Further explanation of this
sample will be provided at the beginning of the
- Label a series of 16 x 125 mm screw-cap
test tubes as: Blank, STD-5, STD-10, STD-15, Urine, Control.
- Add 2.0 mL of appropriate sample to each
tube, using distilled water for the BLANK. Then add 1.0
mL ammonia buffer to each of the six tubes and mix.
- Add 4.0 mL of extraction solvent to all
tubes, caps, and mix by inversion for 5-10 minutes.
- Centrifuge the tubes for 2 minutes at 500
RPM using a clinical centrifuge. Use a setting of 8 on
- Aspirate the (UPPER) aqueous layers
and then decant each (LOWER) organic layer into a
clean, correspondingly labeled 16 x 125 mm screw-cap
- Add 4.0 mL of 0.1 N H2SO4
to each tube. Cap tubes, and mix by inversion for 5-10
- Centrifuge tubes for 2 minutes and 1500
- Transfer each (UPPER) aqueous layer
to a labeled test tube by means of a Pasteur pipet.
- Start the spectrofluorometer, following
the instructions as listed in the instruction manual.
Consult the instruction manual for guidelines on setting
slit widths and scan speeds.
- Measure the spectra as listed below;
- Emission spectra of quinine in 0.1 N H2SO4:
Use 10 mg/L standard as originally prepared. Set the
excitation wavelength to 350 nm and scan from 200 nm to
750 nm using the emission monochromator.
- Excitation Spectra of quinine in 0.1 N H2SO4:
Using the same solution, and an emission wavelength near
the maximum found from the above step, scan the
excitation wavelength from 200-340 nm.
- Set the excitation wavelength to 350 nm
and perform an emission scan of blank solution from
- Set the excitation wavelength to 350 nm.
Set emission wavelength to the optimum wavelength found
from 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 from 300-600 nm.
- With controls adjusted, run emission scans
on all remaining solutions including control and unknown.
- Measure the fluorescence signal of all
solutions and prepare table as shown below:
- Plot corrected fluorescence signal as a
function of concentration (mg/L) and determine
concentration of control and unknown in mg/L.
- Identify; a) Raleigh scattering peak, b)
Raman spectra, and c) second order spectra, in the blank
- Comment on spectra of control and urine
relative to pure standard spectra.
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 of time. One starting point
is that of the ultraviolet absorption spectrum for the compound
in question. The excitation wavelength values are logical
selections. Later, these wavelengths can be verified in the
excitation wavelength scan.
The selection of both excitation and emission
wavelengths involves determining the sensitivity, onset of
quenching, interference, etc. It is appropriate in methodology
set-up to scan the solution thoroughly before deciding on fixed
wavelengths for measurement.
Absorption, emission, fluorescence,
phosphorescence, luminescence, photoluminescence,
chemiluminescence, thermal relaxation, intersystem crossing, and
internal conversion are commonly used terms in spectroscopy.
Define 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 of the kinetics of fluorescence
should be included in all reports. In your conclusions, comment
on the utility and sensitivity of fluorescence. Those of you who
wish to do your formal report on fluorescence spectroscopy should
consider the following ideas.
- Compare the absorbance spectrum with the
fluorescence spectrum. What are the similarities and
differences? Account for these phenomena.
- A discussion on fluorescence quenching
could be given.
- Why do chemicals fluoresce? Do all
chemical species fluoresce?
- What is a Jablonski diagram?