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n te Laboratory
Comparison of Analytical Methods: Direct Emission
versus First-Derivative Fluorometric Methods
for Quinine Determination in Tonic Waters
Siddharth Pandey, Tammie L. Borders, Carmen E. Hernandez, Lindsay E. Roy, Gaddum D. Reddy, Geo
L. Martinez, Autumn Jackson, Guenevere Brown, and William E. Acree, Jr.*
Department of Chemistry, University of North Texas, Denton, TX 76203-5070Fluorescence spectroscopy is an extremely versatile,
sensitive experimental technique used in identification and
quantification of many environmentally important compounds
such as polycyclic aromatic hydrocarbons, polycyclic aromatic
nitrogen heterocycles, and polycyclic aromatic sulfur hetero-
cycles. Through judicious selection of excitation and emission
wavelengths, a single desired fluorophore can often be analyzed
in complex unknown mixtures containing several absorbing
and fluorescing species.
Many laboratory experiments appearing in this Journal
(1-,) and standard laboratory manuals (e.g., ref 10) have
involved determination of analyte concentrations by fluoro-
metric methods. Published methods assume that the observed
emission intensity, F, is
F= KC (1)
directly proportional to the molar concentration of the
analyte. The proportionality constant, K, depends upon
the quantum efficiency (quantum yield) of the fluorescence
process, the response of the photodetector at the emission
wavelength, and the molar extinction coefficient, which
remain constant during any given chemical analysis at fixed
excitation and emission wavelengths. Analyte concentrations
are determined from a working-curve plot of the measured
fluorescence intensity versus the known molar concentrations
of the standard solutions.
The aforementioned experimental methods introduce
students to fluorescence instrumentation. However, the data
analysis will appear rather trivial if UV-vis spectrophotometric,
flame emission, or AA analysis has already been performed. Most
instrumental analysis textbooks (11-14) discuss absorption spec-
troscopy and applications of the Beer-Lambert law one or two
chapters before presenting fluorescence and phosphorescence.
We have found it possible to modernize our existing fluo-
rometric laboratory experiment involving the determination
of quinine in tonic waters by statistically comparing values
determined from direct emission and first-derivative fluoro-
metric methods. Recent review articles (15-20), written in
several different languages, have cited numerous examples of
the application of derivative spectroscopy to the analysis of
food, clinical, pharmaceutical, biomedical, and environmental
samples. For the most part, published applications utilize
either the first or second derivative. Third and higher-order
derivatives have been successfully used in select occasions. The
first-derivative spectrofluorometric method is relatively
*Corresponding author. Email: acree@unt.edu; Fax: 940/565-
4318.straightforward and will be discussed in terms of an unknown
tonic water sample containing quinine. The measured emission
intensity is given by eq 1. Differentiation of the solution
fluorescence emission with respect to the emission wave-
length, Xem, yields the following mathematical expression:
dF/d Xem = (dK/d Xem) quinine (2)
For solutions that contain only a single fluorophore, the first
derivative corresponds to the gradient dF/d Xem of the fluo-
rescence emission envelope and for each well-resolved band
features only a maximum and trough. The vertical distance is
the amplitude, which is directly proportional to the analyte
concentration at each wavelength provided that eq 1 is
obeyed. The proportionality constant, dK/dXem, is obtained
from linear least-squares analysis of the first-derivative
spectrofluorometric data for standard solutions of known
quinine concentration.
The first-derivative method is identical in concept to the
more conventional fluorescence method based upon eq 1,
except that first-derivative spectra are used in the data treatment.
Many scanning spectrofluorometers have built-in software for
displaying derivative spectra. We have found that it requires
very little additional laboratory time to record first-derivative
spectra as part of the experimental laboratory measurements.
Students are instructed to compare their quinine concentrations
calculated from the direct emission intensities to values obtained
from the first-derivative spectra (both positive and negative
slopes) to ascertain if there is a significant difference in the
analytical methods. Values from the entire class are pooled to
increase the number of data points for the statistical treatment.
The statistical treatment is discussed in most standard ana-
lytical textbooks (21-23). Rarely are undergraduate students
afforded the opportunity to actually apply the treatment to
their experimental data. Such analysis leads into a discussion
of factors that are considered in analytical method selection.
The selection of an appropriate analytical method is a decision
that practicing analytical chemists encounter daily.
Experimental Measurements
The experimental work can be completed easily in a 3-
hour laboratory period. We suggest that students work in
groups of two to reduce the time needed to prepare solutions.
Each group is given 50 mL of tonic water for analysis and
told that the sample must be diluted with 0.05 M H2SO4 in
order to have the measured emission intensity fall in the linear
region of the working curve. A 20-fold dilution (5 mL aliquotJChemEd.chem.wisc.edu - Vol. 76 No. 1 January 1999 - Journal of Chemical Education
85
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Pandey, Siddharth; Borders, Tammie L.; Hernández, Carmen E.; Roy, Lindsay Elizabeth; Reddy, Gaddum D.; Martinez, Geo L. et al. Comparison of Analytical Methods: Direct Emission versus First-Derivative Fluorometric Methods for Quinine Determination in Tonic Waters, article, January 1, 1999; [Washington, D.C.]. (https://digital.library.unt.edu/ark:/67531/metadc674086/m1/1/: accessed April 23, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT College of Arts and Sciences.