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Received: 3 November 2024
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Accepted: 25 December 2024
Published: 6 January 2025
Citation: Harcher, A.; Ricard, C.;
Connolly, D.; Gibbs, I.; Shaw,J.; Butler,
J.; Perschbacher, J.; Replogle, L.; Eide,
M.; Grissom, M.; et al. Fluorescence
Analysis of Quinine in Commercial
Tonic Waters. Methods Protoc. 2025,8, 5.
https://doi.org/10.3390/
mps8010005
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Article
Fluorescence Analysis of Quinine in Commercial Tonic Waters
Artturi Harcher, Connor Ricard, Danielle Connolly, Isabel Gibbs, Jarve Shaw, Jillian Butler, Julia Perschbacher,
Lindsay Replogle, Michaela Eide, Morgan Grissom, Oliver O’Neal, Quan Nguyen, Van Hac Nguyen,
Michael Hunnicutt, Roaa Mahmoud and Soma Dhakal *
Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, Richmond, VA 23284, USA
*Correspondence: sndhakal@vcu.edu
Abstract: Quinine is known for treating malaria, muscle cramps, and, more recently, has
been used as an additive in tonic water due to its bitter taste. However, it was shown that
excessive consumption of quinine can have severe side effects on health. In this work, we
utilized fluorescence spectroscopy to measure the concentration of quinine in commercial
tonic water samples. An external standard method was used to calculate the concentrations
of quinine in two commercially available tonic water brands, namely Canada Dry and
Schweppes, and compare them to the maximum allowable concentration of quinine in
beverages. Upon analysis of the data collected by five different groups, the levels of quinine
were found to be above the average concentration in most commercial tonic water samples,
but below the maximum permitted concentration. Moreover, the five replicate sets of data
demonstrated high reproducibility of the method employed in this study. The simple yet
instructive protocol that we developed can be adapted to determine the concentration of
other fluorescent compounds in foods and beverages. Further, the presented method and
detailed protocol can be easily adopted for undergraduate labs and in chemical education.
Keywords: quinine; fluorescence spectroscopy; quantitative analysis; detection; food and
drug chemistry
1. Introduction
Quinine is historically known as an antimalarial drug used to treat the mosquito-
borne disease malaria [
1
]. Due to its bitter taste, it is also used as a flavoring agent in
soft drinks, such as tonic water [
2
]. However, despite its therapeutic applications, the
U.S. Food and Drug Administration (FDA) has limited the maximum allowable amount
of quinine in tonic water to 83 ppm [
3
] due to the adverse effects that quinine can cause
if consumed in high doses, such as nausea, vomiting, kidney injury, and disseminated
intravascular coagulation [
4
–
6
]. Therefore, it is important to employ analytical methods to
check the levels of quinine in food products and beverages to ensure they meet regulatory
requirements. This work serves as a good example of how to monitor fluorescent analytes
in drinks. Structurally, quinine is characterized as a fluorescent molecule with analytically
useful excitation wavelengths at 250 and 350 nm and a fluorescence emission wavelength at
450 nm in dilute acidic solutions [
4
,
7
]. Compared to other fluorescent molecules examined,
such as pyrene in hexane and 2-acetylnaphthalene in water, quinine is fairly stable in acidic
solutions and is not highly susceptible to quenching by oxygen [
8
]. Moreover, the ease
of handling and straightforward sample preparation of quinine makes its analysis more
feasible under a relatively safe laboratory environment.
Several instrumental techniques have been reported for the detection and determi-
nation of quinine in biological and food samples. For example, Castro et al. developed a
Methods Protoc. 2025,8, 5 https://doi.org/10.3390/mps8010005
Methods Protoc. 2025,8, 5 2 of 12
fourth-order derivative spectrophotometric method to determine quinine in soft drinks
in the presence of other additives [
9
]. Other studies used atomic absorption spectrome-
try [
10
], flow-injection chemiluminescence [
11
,
12
], gas chromatography [
13
], and liquid
chromatography [
14
,
15
]. Although these methods are sensitive and eliminate interference
from the sample matrix, they are generally time-consuming and require complicated sam-
ple preparation steps and data analysis procedures. Therefore, in this work, we developed
a simplified and detailed experimental protocol using a widely employed and known
technique, fluorescence spectroscopy, to determine quinine concentration in carbonated
beverages (tonic water samples). A few studies have been reported in the literature for the
detection of quinine using fluorescence-based assays [
16
–
19
]. For example, de Souza et al.
devised an image-based fluorometric system equipped with an LED camera and a digital
microscope to detect quinine in commercial samples [
18
]. Although high quantification
limits were achievable using this fluorometric assay, the proposed method is relatively
complex involving the use of mathematical models and post-analysis software for data ac-
quisition and processing. Similarly, while Tsaftari and coworkers presented a paper-based
fluorometric technique requiring minimal use of instrumentation for the rapid detection of
quinine [
19
], the paper-based platforms are typically fragile and require careful handling
to ensure reproducibility. Overall, although these publications are excellent resources for
the analysis of quinine, they are not particularly written as method papers and are not
readily followed by less-experienced personnel such as undergraduates. To fill this gap,
this manuscript is designed to be a more complete reference guide for quinine analysis
in tonic waters targeted toward both students and instructors primarily in the fields of
analytical chemistry, physical chemistry, spectroscopy, and chemical education. In fact, this
protocol is a result of systematic experiments performed by several groups of students for
their senior capstone project, where a total of 13 students distributed in five groups (2 or
3 students each) took part in the experiments.
Fluorescence spectroscopy is a widely used analytical technique that has many practi-
cal applications due to its high analytical sensitivity [
20
,
21
]. For example, a spectrofluorom-
eter is typically used for inorganic chemistry applications such as for the determination
of chromium and manganese in steel or aluminum in alloys [
22
]. It is also commonly
applied in the identification and quantification of organic compounds such as polycyclic
aromatic hydrocarbons [
23
]. In principle, fluorescence is the emission of a molecule from
its singlet excited electronic state to its ground state upon the absorption of UV or visible
radiation [
20
,
22
]. Because of the direct relationship between the intensity of fluorescence
and the concentration of an analyte in dilute solutions, fluorescence spectroscopy has
been used to quantify quinine in tonic water samples. For example, several other studies
have reported the use of custom-designed miniaturized fluorimeters for the detection and
quantification of various compounds including quinine [
24
–
28
]. Compared to the previous
studies, this work presents a systematically laid out protocol to measure the concentration
of quinine in commercial tonic water samples using a spectrofluorometer while following
strictly detailed step-by-step experimental and technical guidelines as provided in the
Supplementary Information. In addition to determining the concentration of quinine in
two tonic water brands (Canada Dry and Schweppes), the reproducibility and overall
experimental precision of the method were assessed in this activity using five different sets
of data collected independently. Overall, the level of quinine in tonic water was found to be
within the acceptable range set by the FDA [
3
], and the method applied here was proven to
be reliable given the high reproducibility of the data.
Methods Protoc. 2025,8, 5 3 of 12
2. Materials and Equipment
Quinine sulfate dihydrate (QSD) was obtained from Mallinckrodt Pharmaceuticals,
Cruiserath, Dublin, Ireland. The two one-liter tonic water bottles (Canada Dry and
Schweppes) were purchased from commercial stores (e.g., Kroger, Richmond, VA, USA).
Concentrated sulfuric acid (2.0 N, N stands for Normality, see Supplementary Information)
and 18 M
Ω
deionized water were used to prepare the 0.05 M sulfuric acid solution (refer to
Supplementary Information for more details on sample preparation and concentration cal-
culations).
3. Hazards and Safety Precautions
Standard laboratory safety measures should be followed when handling hazardous
chemicals. Quinine sulfate dihydrate (QSD) is a skin, eye, and respiratory irritant if inhaled.
Precautions for QSD include wearing PPE, handling in a ventilated area, and avoiding
inhalation of dust that can form.
4. Experimental Procedure
4.1. Sample Preparation
A 0.05 M sulfuric acid solution was prepared from the 2.0 N concentrated sulfuric
acid solution and diluted with 18 M
Ω
deionized water (see the Chemicals and Samples
section in the Supplementary Information). The prepared 0.05 M sulfuric acid solution was
used as the diluent for all the subsequent standards and samples. A 1000 ppm quinine
stock solution was prepared by weighing ~121 mg of QSD (structure and molecular weight
information can be found in the Supplementary Information) and transferring it to a
100 mL volumetric flask, followed by dilution with 0.05 sulfuric acid. A 100 ppm quinine
standard solution was prepared from the 1000 ppm solution, followed by further dilution
to a 1 ppm quinine standard solution. Five calibration standards (0.2–5.0 ppm) were also
prepared from the 100 ppm quinine standard in 25 mL volumetric flasks (see Table S1). The
commercial quinine tonic water samples were prepared by delivering 2 mL of the tonic
water sample into a 50 mL volumetric flask and diluting it with 0.05 M sulfuric acid (more
details can be found in the Supplementary Information).
4.2. Generating Excitation and Emission Spectra
Excitation and emission spectra were collected for the 1 ppm quinine standard solution
using the scan mode of the Agilent Varian-Cary eclipse fluorescence spectrometer with
preinstalled scan and advanced reads programs for spectra recording and analysis (check
the Supplementary Information for more details). Briefly, a quartz cuvette was filled
with the 1 ppm solution and placed into the sample compartment of the spectrometer for
analysis. To collect the excitation spectrum, the excitation monochromator was scanned over
a wavelength range of 190–440 nm, while the emission monochromator was set to 450 nm.
Similarly, two emission spectra were obtained by scanning the emission monochromator
from 360 to 600 nm and setting the excitation either to 250 nm or 350 nm. For each spectrum,
the slit widths of the monochromators were set to 5.0 nm and the photomultiplier tube
(PMT) detector voltage was set to 600 V (Table S2).
4.3. Photomultiplier Tube (PMT) Voltage and Emission Intensity
The effect of PMT voltage on fluorescence intensity was assessed using the 1 ppm
quinine solution and the advanced reads mode of the instrument software (refer to the
Supplementary Information for more details). Briefly, the fluorescence intensity was
measured as a function of PMT voltage, which varied from 400 to 725 V. The excitation and
Methods Protoc. 2025,8, 5 4 of 12
emission monochromators were set to 350 nm and 450 nm, respectively, and the slit widths
were held constant at 5.0 nm.
4.4. Monochromator Slit Width and Emission Intensity
The effect of the excitation and emission monochromator slit widths on fluorescence
intensity were independently analyzed using the 1 ppm quinine standard solution and
the advanced reads mode of the instrument (see the PMT and Slit Width section in the
Supplementary Information). The first part consisted of varying the excitation slit width
from 1.5 to 20 nm while keeping the emission slit width at 2.5 nm, and the second part
consisted of varying the emission slit width (1.5–20 nm) and maintaining the excitation slit
at 2.5 nm. In both parts of the experiment, the excitation and emission monochromator
wavelengths were set to 250 nm and 350 nm, respectively, and the PMT voltage was set
to 600 V.
4.5. Statistical Analysis
All the experiments described here were performed by 5 groups of individuals
(2–3 students each group). Each group provided one set of data for all experiments,
and the mean value and standard deviation (S.D., shown as error bars) were calculated
from five data sets (n= 5). All calculations were performed in Excel. It is important to note
that the data were collected over the course of about 5 weeks (one group experimented per
week); therefore, slight distribution observed in the datasets may be attributed to variations
in instrument performance and/or sample preparation by different groups.
5. Results and Discussion
The first part of the experimental protocol consisted of a set of experiments performed
to illustrate the effects of different instrumental parameters on the intensity of fluorescence
emission (see the Supplementary Information for a step-by-step experimental protocol).
Through these sets of experiments, the reproducibility of the instrument was also inves-
tigated across five different data sets collected independently. First, prior to recording
fluorescence emission, excitation and emission spectra for the 1 ppm quinine solution were
collected to select the wavelengths (
λmax
) for the subsequent experiments. The spectra from
all five independent data sets are shown in Figure 1.
As depicted in Figure 1, the
λmax
values did not vary significantly for the S
1
and S
2
peaks in the excitation spectra across the five sets of experiments. The mean wavelengths
and standard deviations for S
1
and S
2
were 345
±
1 nm and 250
±
1 nm, respectively. In
addition, the emission wavelengths were similar to one another (455
±
2 nm for E
1
and
456
±
2 nm for E
2
). The observed
λmax
values from the absorption and emission spectra of
quinine are commensurate with the previously reported wavelengths of 347 nm and 452 nm
for S
1
and E, respectively [
18
]. The slightly higher standard deviation for the emission
spectra is expected because of the different ways an excited electron can dissipate its energy
within an electronic state (i.e., the presence of vibrational states within the ground electronic
state, resulting in different emission wavelengths) [
8
,
10
]. Furthermore, variations could
be due to instrument fluctuations or less successful nonradiative processes resulting in
slightly different emission wavelengths. Unlike the
λmax
values, the maximum intensity
values for each listed wavelength were found to be less consistent. The mean maximum
intensity values for the S
1
, S
2
, and E
1
were 138
±
13, 506
±
50, and 133
±
12, respectively.
E
2
(emission from excitation at 250 nm) was excluded from this analysis as it is not the
λem
chosen for the following experiments; thus, its intensity fluctuations are not as relevant and
do not factor into the variations seen in the calculated quinine levels. As can be seen from
the standard deviations of intensity, the maximum intensity values varied significantly
Methods Protoc. 2025,8, 5 5 of 12
between the five groups. Variations in fluorescence intensity are likely due to human
errors (e.g., errors in weighing, pipetting, etc.) which can vary slightly from one group to
another, resulting in different prepared concentrations of quinine. Moreover, quinine stock
solutions had to be freshly prepared several times during the experiments, which might
have resulted in slight differences in the final quinine concentrations. Notably, solutions at
high concentrations can have an internal screening effect, leading to lower fluorescence
intensities than expected.
Methods Protoc. 2025, 8, 5 4 of 12
Supplementary Information for more details). Briefly, the fluorescence intensity was
measured as a function of PMT voltage, which varied from 400 to 725 V. The excitation
and emission monochromators were set to 350 nm and 450 nm, respectively, and the slit
widths were held constant at 5.0 nm.
4.4. Monochromator Slit Width and Emission Intensity
The effect of the excitation and emission monochromator slit widths on fluorescence
intensity were independently analyzed using the 1 ppm quinine standard solution and
the advanced reads mode of the instrument (see the PMT and Slit Width section in the
Supplementary Information). The first part consisted of varying the excitation slit width
from 1.5 to 20 nm while keeping the emission slit width at 2.5 nm, and the second part
consisted of varying the emission slit width (1.5–20 nm) and maintaining the excitation
slit at 2.5 nm. In both parts of the experiment, the excitation and emission monochromator
wavelengths were set to 250 nm and 350 nm, respectively, and the PMT voltage was set
to 600 V.
4.5. Statistical Analysis
All the experiments described here were performed by 5 groups of individuals (2–3
students each group). Each group provided one set of data for all experiments, and the
mean value and standard deviation (S.D., shown as error bars) were calculated from five
data sets (n = 5). All calculations were performed in Excel. It is important to note that the
data were collected over the course of about 5 weeks (one group experimented per week);
therefore, slight distribution observed in the datasets may be aributed to variations in
instrument performance and/or sample preparation by different groups.
5. Results and Discussion
The first part of the experimental protocol consisted of a set of experiments per-
formed to illustrate the effects of different instrumental parameters on the intensity of
fluorescence emission (see the Supplementary Information for a step-by-step experi-
mental protocol). Through these sets of experiments, the reproducibility of the instrument
was also investigated across five different data sets collected independently. First, prior
to recording fluorescence emission, excitation and emission spectra for the 1 ppm quinine
solution were collected to select the wavelengths (λmax) for the subsequent experiments.
The spectra from all five independent data sets are shown in Figure 1.
Figure 1. Excitation and emission spectra of the 1 ppm quinine standard solution. The spectra
collected, one excitation and two emissions, are color-coded to designate the different data sets.
Spectra were obtained with monochromator slit widths of 5.0 nm and a PMT voltage of 600 V. The
λmax
values for the first excited singlet state (S
1
), second excited singlet state (S
2
), first emission (E
1
),
and second emission (E
2
) are listed. Five replicates were recorded for excitation and emission spectra
(n= 5). Spectra were collected at room temperature.
The next portion of this study comprised varying the PMT voltage and measuring
the fluorescence intensity. Intensities were measured at PMT voltages of 400–850 V using
the 1 ppm quinine standard solution. The rationale behind this experiment is to examine
the effect of the PMT voltage on the sensitivity of the instrument. Figure 2illustrates
the relationship between the PMT voltage and the measured intensity. It is evident from
the plot in Figure 2A that the measured fluorescence intensity increased nonlinearly with
increasing PMT voltage. This is expected because according to the photoelectric effect, as
the PMT voltage increases, the voltage difference between the dynodes (i.e., electrodes)
increases and, thus, the signal is exponentially amplified [
29
]. The correlation coefficient
values retrieved from the best-fit curves for each group indicate a good correlation between
the fluorescence intensity and PMT voltage. However, it is important to note that the data
points corresponding to 800–850 V were omitted from the analysis as the fluorescence
signal was saturated at high PMT voltages (the full curve including the signal saturation is
provided in Figure S1).
Methods Protoc. 2025,8, 5 6 of 12
Methods Protoc. 2025, 8, 5 6 of 12
Figure 2. (A) Plot of fluorescence intensity vs. PMT voltage for five different data sets. Data points
were best fit with the power function (second order), and the corresponding correlation coefficient
R2 values are provided. (B) Plot of the mean fluorescence intensity and PMT voltage. The mean
intensity was calculated by averaging out the five intensity values at each voltage, and the corre-
sponding standard deviation is represented by the error bars (n = 5). The correlation coefficient for
the best-fit line is shown on the plot.
Further examination of the reproducibility of fluorescence intensity at each PMT volt-
age revealed more variations at higher PMT voltages, which is obvious from the larger
error bars (standard deviations) in Figure 2B. This observation is likely due to hiing the
near-saturation limit of the PMT detector at higher voltages, which could affect the detec-
tion response and the signal reading. Nonetheless, the correlation coefficient obtained for
the plot of mean fluorescence intensity and PMT voltage is comparable to the correlation
coefficients obtained by the individual groups. Again, this illustrates a good correlation
between the measured fluorescence intensity and PMT voltage. Overall, the data is more
or less consistent at low-mid PMT voltages, which demonstrates good reproducibility of
the data and a uniform instrument response.
Using the same approach, the 1 ppm quinine standard solution was also used to
measure the fluorescence intensity while systematically varying excitation monochroma-
tor slit width. The intensity was measured at the slit widths of 1.5, 2.5, 5, 10, and 20 nm.
Figure 3 shows the change in fluorescence intensity as a function of excitation mono-
chromator slit width. As shown by the plot, the intensity increased linearly due to increas-
ing excitation slit width. This direct linear relationship between intensity and excitation
slit is illustrated by Equation (1) [29], where IF(λ) is emission intensity, Po is the power of
the incident radiation, ℰ() is the molar absorptivity at the excitation wavelength, c is
the concentration, l is the pathlength, QF is the quantum yield, and k is the ratio of ab-
sorbed to emied photons.
𝐼()=𝑃
(2.303ℰ()𝑐𝑙)𝑄𝑘 (1)
The rationale is that increasing the excitation slit width increases the amount of inci-
dent light and, thus, the number of photons that can be absorbed by the sample. This
increases k, i.e., the ratio of absorbed to emied photons, which increases the fluorescence
intensity. As Equation (1) shows, the fluorescence intensity and ratio k are directly pro-
portional, indicating a direct and linear relationship, provided other variables are con-
stant. Variations in the measured intensity were identified among the independent data
sets, particularly at larger slit widths (Figure 3B). Based on Equation (1), variations could
be due to differences in the quinine standard concentration, which are more prominent at
larger slit widths (i.e., higher k), or due to instrument fluctuations in the power of the
incident radiation (𝑃
). It is important to note that changing the excitation monochromator
Figure 2. (A) Plot of fluorescence intensity vs. PMT voltage for five different data sets. Data points
were best fit with the power function (second order), and the corresponding correlation coefficient R
2
values are provided. (B) Plot of the mean fluorescence intensity and PMT voltage. The mean intensity
was calculated by averaging out the five intensity values at each voltage, and the corresponding
standard deviation is represented by the error bars (n= 5). The correlation coefficient for the best-fit
line is shown on the plot.
Further examination of the reproducibility of fluorescence intensity at each PMT
voltage revealed more variations at higher PMT voltages, which is obvious from the larger
error bars (standard deviations) in Figure 2B. This observation is likely due to hitting
the near-saturation limit of the PMT detector at higher voltages, which could affect the
detection response and the signal reading. Nonetheless, the correlation coefficient obtained
for the plot of mean fluorescence intensity and PMT voltage is comparable to the correlation
coefficients obtained by the individual groups. Again, this illustrates a good correlation
between the measured fluorescence intensity and PMT voltage. Overall, the data is more or
less consistent at low-mid PMT voltages, which demonstrates good reproducibility of the
data and a uniform instrument response.
Using the same approach, the 1 ppm quinine standard solution was also used to
measure the fluorescence intensity while systematically varying excitation monochromator
slit width. The intensity was measured at the slit widths of 1.5, 2.5, 5, 10, and 20 nm. Figure 3
shows the change in fluorescence intensity as a function of excitation monochromator slit
width. As shown by the plot, the intensity increased linearly due to increasing excitation slit
width. This direct linear relationship between intensity and excitation slit is illustrated by
Equation (1) [
29
], where I
F(λ)
is emission intensity, P
o
is the power of the incident radiation,
E(λexci t)
is the molar absorptivity at the excitation wavelength, cis the concentration, lis
the pathlength, Q
F
is the quantum yield, and kis the ratio of absorbed to emitted photons.
IF(λ)=Po2.303E(λexcit)clQFk(1)
The rationale is that increasing the excitation slit width increases the amount of incident
light and, thus, the number of photons that can be absorbed by the sample. This increases
k, i.e., the ratio of absorbed to emitted photons, which increases the fluorescence intensity.
As Equation (1) shows, the fluorescence intensity and ratio kare directly proportional,
indicating a direct and linear relationship, provided other variables are constant. Variations
in the measured intensity were identified among the independent data sets, particularly at
larger slit widths (Figure 3B). Based on Equation (1), variations could be due to differences
in the quinine standard concentration, which are more prominent at larger slit widths
(i.e., higher k), or due to instrument fluctuations in the power of the incident radiation
(
Po)
. It is important to note that changing the excitation monochromator slit width should
Methods Protoc. 2025,8, 5 7 of 12
not, in principle, affect the emission wavelength resolution, provided that the emission
monochromator slit width is held constant.
Methods Protoc. 2025, 8, 5 7 of 12
slit width should not, in principle, affect the emission wavelength resolution, provided
that the emission monochromator slit width is held constant.
Figure 3. (A) Plot of fluorescence intensity vs. excitation monochromator slit width for the five
groups. The linear best-fit lines, linear regression equations, and correlation coefficients are dis-
played for each group. (B) Plot of the mean fluorescence intensity as a function of excitation slit
width. Error bars represent standard deviations from five datasets (n = 5). Data were collected at a
PMT voltage of 600 V with an emission monochromator slit width of 5 nm.
The last portion relating to instrumental parameters involved recording the fluores-
cence intensity at different emission monochromator slit widths. As in the excitation slit
scenario, increasing the emission monochromator slit width is expected to increase the
fluorescence intensity. This is due to the increased amount of emied light permied to
reach the PMT detector. However, as shown in Figure 4A, which depicts the plot of the
square root of intensity against emission slit width, the relationship was best represented
by a quadratic (polynomial) rather than a linear function. This implies that the fluores-
cence signal is nonlinearly affected by the emission monochromator slit width.
Figure 4. (A) Plot of the square root of fluorescence intensity vs. emission monochromator slit width
for the five groups. The polynomial best-fit lines, quadratic equations, and correlation coefficients
are displayed for each group. (B) Plot of the mean square root of intensity as a function of emission
slit width. Error bars denote standard deviations from five datasets (n = 5). Data were collected at a
PMT voltage of 600 V with an excitation monochromator slit width of 5 nm.
Figure 3. (A) Plot of fluorescence intensity vs. excitation monochromator slit width for the five groups.
The linear best-fit lines, linear regression equations, and correlation coefficients are displayed for each
group. (B) Plot of the mean fluorescence intensity as a function of excitation slit width. Error bars
represent standard deviations from five datasets (n= 5). Data were collected at a PMT voltage of
600 V with an emission monochromator slit width of 5 nm.
The last portion relating to instrumental parameters involved recording the fluores-
cence intensity at different emission monochromator slit widths. As in the excitation slit
scenario, increasing the emission monochromator slit width is expected to increase the
fluorescence intensity. This is due to the increased amount of emitted light permitted to
reach the PMT detector. However, as shown in Figure 4A, which depicts the plot of the
square root of intensity against emission slit width, the relationship was best represented
by a quadratic (polynomial) rather than a linear function. This implies that the fluorescence
signal is nonlinearly affected by the emission monochromator slit width.
Hence, we plotted the square root of the intensity as a function of the emission slit to
highlight the difference in the instrument response to variations in excitation and emission
slits. Unlike Figure 3, minimal variation in the measured intensity was noted between the
groups, as shown in Figure 4B. This could be due to a smaller number of instrumental
variables that contribute to variations in emission intensity given a constant excitation
slit width. However, unlike excitation, increasing the emission monochromator slit width
reduced the emission wavelength resolution.
After evaluating the instrument response and validating the reproducibility of the
data, the concentration of quinine in commercial tonic water samples was determined and
the results from the five groups were compiled and analyzed (check the External Calibra-
tion Standards section in the Supplementary Information). To determine the unknown
concentration of quinine, five calibration standards with known concentrations of quinine
were prepared and their fluorescence intensity was sequentially measured starting with
the lowest concentration (refer to Table S3 for instrumental settings).
Figure 5depicts two plots of fluorescence intensity as a function of quinine standard
concentration in ppm. The resulting calibration curves demonstrated good linearity and
high correlation coefficient values. Figure 5A shows the overlay of five calibration curves
using the external standard method. A slight deviation in the measured intensity can be
seen at higher quinine concentrations, which could be due to human error while preparing
the standards or instrumental errors as described earlier. To cross-compare, we included
Methods Protoc. 2025,8, 5 8 of 12
another set of calibration data collected by an independent group of individuals shown in
the Supplementary Information (Figure S2). Comparing the two sets of calibration curves,
we can conclude that the data are reproducible with a good linearity (R
2
> 0.99). The slight
differences among experiments can be attributed to variations in sample preparations.
Methods Protoc. 2025, 8, 5 7 of 12
slit width should not, in principle, affect the emission wavelength resolution, provided
that the emission monochromator slit width is held constant.
Figure 3. (A) Plot of fluorescence intensity vs. excitation monochromator slit width for the five
groups. The linear best-fit lines, linear regression equations, and correlation coefficients are dis-
played for each group. (B) Plot of the mean fluorescence intensity as a function of excitation slit
width. Error bars represent standard deviations from five datasets (n = 5). Data were collected at a
PMT voltage of 600 V with an emission monochromator slit width of 5 nm.
The last portion relating to instrumental parameters involved recording the fluores-
cence intensity at different emission monochromator slit widths. As in the excitation slit
scenario, increasing the emission monochromator slit width is expected to increase the
fluorescence intensity. This is due to the increased amount of emied light permied to
reach the PMT detector. However, as shown in Figure 4A, which depicts the plot of the
square root of intensity against emission slit width, the relationship was best represented
by a quadratic (polynomial) rather than a linear function. This implies that the fluores-
cence signal is nonlinearly affected by the emission monochromator slit width.
Figure 4. (A) Plot of the square root of fluorescence intensity vs. emission monochromator slit width
for the five groups. The polynomial best-fit lines, quadratic equations, and correlation coefficients
are displayed for each group. (B) Plot of the mean square root of intensity as a function of emission
slit width. Error bars denote standard deviations from five datasets (n = 5). Data were collected at a
PMT voltage of 600 V with an excitation monochromator slit width of 5 nm.
Figure 4. (A) Plot of the square root of fluorescence intensity vs. emission monochromator slit width
for the five groups. The polynomial best-fit lines, quadratic equations, and correlation coefficients are
displayed for each group. (B) Plot of the mean square root of intensity as a function of emission slit
width. Error bars denote standard deviations from five datasets (n= 5). Data were collected at a PMT
voltage of 600 V with an excitation monochromator slit width of 5 nm.
Using the linear regression equations determined from the calibration curves, the
concentration of quinine in the tonic water samples (Canada Dry and Schweppes) was
calculated. For the external standard method, the individual results from the five groups are
shown in Table 1along with the mean quinine concentration and standard deviations. As
described in the experimental section and SI, the tonic water samples were diluted before
recording the fluorescence measurements, and, thus, there are two quinine concentrations
denoted as diluted and undiluted, as shown in Table 1. The undiluted concentration repre-
sents the actual quinine concentration in the tonic water sample considering the dilution
factor (25-fold). As illustrated in Table 1, there is some variation in the calculated quinine
concentrations among the five groups, especially for the Canada Dry tonic water sample.
This is to be expected considering that the measurements were taken at different
times/days and the possibility of minor variations in sample preparation. However, the
results are consistent as the standard deviations are relatively small. The mean concen-
trations of the two tonic water samples were determined to be approximately 65 ppm
for both samples, which is slightly higher than the average concentration range of qui-
nine in commercial tonic water (~25–60 ppm) [
4
]. However, the quinine concentration of
~65 ppm is well below the FDA requirements of 83 ppm or less for quinine [
3
,
24
]. Fitting
the calibration data also allowed us to estimate the limit of detection (LOD) of quinine,
which was 0.2 ppm.
In addition, the quinine concentration was also determined using an internal standard
approach as a complementary method (Figure 5B). The standard addition allows for the
measuring of fluorescence in the presence of any interferences caused by the sample matrix.
In this experiment, the Canada Dry sample was used as an example and the quinine
quantity was determined in the sample (using two replicates) and cross-compared with the
result from the external standard method. Briefly, the quinine concentration was calculated
using the extrapolation of data, in which the x-intercept represents the concentration of the
Methods Protoc. 2025,8, 5 9 of 12
unknown quinine sample [Q]
x
(Figure 5B). Given the 25-fold dilution, the extrapolated
concentration of quinine was multiplied by the dilution factor to calculate the actual
(undiluted) concentration, which was found to be ~57
±
26 ppm. This result is comparable
to the concentration in the Canada Dry sample (~65
±
4 ppm) determined using the
external calibration method (Figure 5A). The lower quinine concentration and larger error
determined by the internal standard method may be attributed to the matrix effect. Further,
the day-to-day reproducibility of the method is inclusive, given that the data were collected
on different days by independent groups of students. The consistent fluorescence data
and calculated quinine levels confirm the reproducibility of the method over the course of
days/weeks. Taken together, the quinine concentration is consistent with the concentration
ranges of 57–80 ppm, 48–67 ppm, and 62–67 ppm in different tonic water samples reported
previously using a PerkinElmer FL6500 fluorescence spectrometer, a LS-50B luminescence
spectrometer, and a reverse-phase HPLC method, respectively [2,16,18].
Methods Protoc. 2025, 8, 5 8 of 12
Hence, we ploed the square root of the intensity as a function of the emission slit to
highlight the difference in the instrument response to variations in excitation and emission
slits. Unlike Figure 3, minimal variation in the measured intensity was noted between the
groups, as shown in Figures 4B. This could be due to a smaller number of instrumental
variables that contribute to variations in emission intensity given a constant excitation slit
width. However, unlike excitation, increasing the emission monochromator slit width re-
duced the emission wavelength resolution.
After evaluating the instrument response and validating the reproducibility of the
data, the concentration of quinine in commercial tonic water samples was determined and
the results from the five groups were compiled and analyzed (check the External Calibra-
tion Standards section in the Supplementary Information). To determine the unknown
concentration of quinine, five calibration standards with known concentrations of quinine
were prepared and their fluorescence intensity was sequentially measured starting with
the lowest concentration (refer to Table S3 for instrumental seings).
Figure 5 depicts two plots of fluorescence intensity as a function of quinine standard
concentration in ppm. The resulting calibration curves demonstrated good linearity and
high correlation coefficient values. Figure 5A shows the overlay of five calibration curves
using the external standard method. A slight deviation in the measured intensity can be
seen at higher quinine concentrations, which could be due to human error while prepar-
ing the standards or instrumental errors as described earlier. To cross-compare, we in-
cluded another set of calibration data collected by an independent group of individuals
shown in the Supplementary Information (Figure S2). Comparing the two sets of calibra-
tion curves, we can conclude that the data are reproducible with a good linearity (R2 >
0.99). The slight differences among experiments can be aributed to variations in sample
preparations.
Figure 5. Calibration curves prepared from the quinine standard solutions using the external (A)
and the internal (B) standard addition methods. The linear best-fit lines, linear regression equations,
and correlation coefficients are displayed for each group in panel A (n = 5). Data were collected at
excitation and emission wavelengths of 350 nm and 450 nm, respectively, with a PMT voltage of 600
V and slit widths set to 5 nm each. The Canada Dry tonic water sample was used for the internal
standard (Si) method in panel B, and the fluorescence intensity is an average of two replicates (n =2).
Standard deviations are represented by the error bars.
Using the linear regression equations determined from the calibration curves, the
concentration of quinine in the tonic water samples (Canada Dry and Schweppes) was
calculated. For the external standard method, the individual results from the five groups
are shown in Table 1 along with the mean quinine concentration and standard deviations.
Figure 5. Calibration curves prepared from the quinine standard solutions using the external (A) and
the internal (B) standard addition methods. The linear best-fit lines, linear regression equations,
and correlation coefficients are displayed for each group in panel A (n= 5). Data were collected at
excitation and emission wavelengths of 350 nm and 450 nm, respectively, with a PMT voltage of
600 V and slit widths set to 5 nm each. The Canada Dry tonic water sample was used for the internal
standard (S
i
) method in panel B, and the fluorescence intensity is an average of two replicates (n=2).
Standard deviations are represented by the error bars.
Furthermore, it is critical to probe the effect of interferences such as pH, food additives,
and/or artificial sweeteners, including citric acid and sugars. Previous studies investigated
the effect of chloride ions and artificial additives (e.g., glucose) as major interferences
and found that chloride ions can cause fluorescence quenching; however, no appreciable
interference from sugar additives was noted [
2
,
4
]. More relevantly, maintaining an acidic
environment is important when using the approach presented here; hence, we sought
to check the effect of pH on the quinine fluorescence intensity, wherein we measured
fluorescence emission as a function of pH (Figure S3). The detailed procedure is outlined
in the Methods section of the Supplementary Information. Briefly, sodium phosphate
buffers were made with a pH range of ~6.0–7.9 to which a constant amount of quinine
(10 ppm) was added before measuring the fluorescence. The results showed that, as the
pH increases, the fluorescence intensity slightly decreases, possibly due to some level of
the deprotonation of quinine (pKa 8.4) when increasing the pH [
30
]. Nonetheless, it is
important to note that all the fluorescence experiments in this activity were performed in
an acidic solution (0.05 M H2SO4) that should keep quinine protonated and fluorescent.
Methods Protoc. 2025,8, 5 10 of 12
Table 1. Measured intensity and calculated concentrations (diluted and undiluted) of quinine in
the two commercial tonic water samples. Results represent the mean intensity, mean diluted, and
undiluted concentration of quinine with standard deviations of the five data sets (n= 5). Data were
acquired at excitation and emission wavelengths of 350 nm and 450 nm, respectively, and a PMT
voltage of 600 V with slit widths set to 5 nm each.
Student Group Tonic Water Intensity at 450 nm Diluted Quinine (ppm) Undiluted Quinine (ppm)
1Sample 1 (Canada Dry) 92.59 ±1.21 2.47 61.76
Sample 2 (Schweppes) 90.72 ±2.10 2.42 60.48
2Sample 1 (Canada Dry) 87.43 ±0.15 2.41 60.23
Sample 2 (Schweppes) 94.96 ±1.61 2.62 65.52
3Sample 1 (Canada Dry) 93.91 ±0.66 2.59 64.67
Sample 2 (Schweppes) 91.85 ±1.99 2.53 63.19
4Sample 1 (Canada Dry) 95.19 ±1.02 2.75 68.78
Sample 2 (Schweppes) 91.66 ±0.33 2.64 66.09
5Sample 1 (Canada Dry) 94.80 ±0.78 2.70 67.57
Sample 2 (Schweppes) 94.31 ±1.35 2.69 67.21
Results: Sample 1 (Canada Dry) 92.78 ±3.16 2.58 ±0.15 64.60 ±3.66
Sample 2 (Schweppes) 92.70 ±1.83 2.58 ±0.11 64.50 ±2.68
6. Conclusions
This work highlights the fundamentals and principles of fluorescence spectroscopy
and its application in the detection and quantitation of analytes. Using this technique,
the effects of instrument parameters on fluorescence intensity were investigated and the
amounts of quinine in commercial tonic water samples were successfully determined. The
results from the five different data sets demonstrated the relationships between instrument
parameters and fluorescence intensity, showing an overall good reproducibility among the
data. Additionally, the calibration curves using quinine standards and the quantitation
of the quinine levels in tonic water samples produced consistent results, with calculated
concentrations that are well below the maximum concentration limit of quinine that is
FDA-approved. Overall, besides the simple and instructive protocol that we developed for
quinine, this facile and low-cost spectroscopic technique may be adopted to determine the
amount of other relevant drugs and food additives. The presented method and detailed
experimental protocol can be further implemented in undergraduate teaching labs and in
chemical education.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/mps8010005/s1, Figure S1: Plot of fluorescence intensity vs PMT
voltage. Data were collected at excitation and emission wavelengths of 350 nm and 450 nm and slit
widths of 5 nm. The signal saturates at PMT voltage of ~800 V and above; Figure S2: Calibration
curves prepared from the quinine standard solutions using the external standard method. The linear
best-fit lines, linear regression equations, and correlation coefficients are displayed for each group.
Data were collected at excitation and emission wavelengths of 350 nm and 450 nm, respectively,
with a PMT voltage of 600 V and slit widths set to 5 nm each. The data presented were collected
by a different group of individuals to supplement the calibration curves in Figure 5A (main text);
Figure S3: pH dependence of quinine fluorescence intensity. Data were collected at excitation and
emission wavelengths of 350 nm and 450 nm, respectively, with a PMT voltage of 600 V and slit
widths set to 5 nm each. The concentration of quinine was 10 ppm in the buffer systems and the
pH was measured after the addition of all components using a pH meter. Error bars represent the
standard deviations from three replicates of intensity measurements; Table S1: External standard
Methods Protoc. 2025,8, 5 11 of 12
quinine solutions for the calibration curve including their respective concentration and volume
transferred; Table S2: Internal standard quinine solutions with their respective concentrations and
volume added; Table S3: Measured amounts of the phosphate buffer components and the respective
final pH of the buffer solutions for the pH dependence test; Table S4: Instrumental parameters and
settings for generating excitation and emission spectra; Table S5: Instrument settings to generate
intensity measurements for the quinine standards and tonic water samples.
Author Contributions: Authors A.H., C.R., D.C., I.G., J.S., J.B., J.P., L.R., M.E., M.G., O.O., Q.N., R.M.
and V.H.N. performed experiments, collected and analyzed data, and contributed to manuscript
writing. R.M. took a lead on plotting data and drafting the manuscript. M.H. provided protocols
for the experiments. S.D. conceived the manuscript idea, guided the project, and contributed to
data analysis and manuscript writing. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Data Availability Statement: The data is available upon reasonable request from the authors.
Acknowledgments: The authors thank Roaa Mahmoud for facilitating a considerable portion of this
work, as well as Alan Branigan and Thomas Cecil for assisting with the successful completion of
the laboratory exercises. The authors would also like to thank Joseph Turner and the Department of
Chemistry at VCU for their instrument and technical support.
Conflicts of Interest: The authors declare no conflicts of interest.
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