The Analysis of the Excitation and Emission Wavelengths of Quinine Containing

Solutions Using Ultraviolet-Visible Absorption and Fluorescence Spectroscopy

Contributors: K. Bender, N. Holzapfel, M. McCarty

Dates of Data Collection: 02/01/2017 and 02/08/2017

Date of Submission: 02/15/2017

Returned Date: 02/17/2017

Resubmission Date: 02/24/2017

Introduction

Before one can begin this experiment one should know some background information

on Quinine and Tonic water and the basics of both of the used instrumental techniques to

better understand the importance and educational facets of this experiment.

Quinine is an alkaloid found in the bark of the cinchona tree indigenous to South

America. The natives used the tree bark to cure fevers and malaria. Quinine has been proven

effective against many forms of malaria, including plasmodium vivax, malariae, and

falciparum. Quinine has been used historically to treat leg cramps [1]. Today Quinine is no

longer approved for prescription purposes but it is still found in small dose, over the counter

items such as tonic water. Larger doses of Quinine have also shown potentially harmful side

effects, including Hepatotoxicity, dizziness, light-headedness, blurred vision, bleeding issues,

and reduction of the number of platelets[2] . People can also have life threatening allergic

reactions to the substance. One of the most common sources of Quinine is in tonic water. Due

to the small amount, there is less risk of side effects. Drinking tonic water has been shown to

have quite a bit of health benefits. Due to the use of Quinine in tonic water, if consumed after

physical activity it can help with muscle cramps and soreness. Tonic water also contains sugar

and has a sweeter taste which people enjoy much more than regular water, so it is more

appealing when one needs to hydrate after a workout. Another practical use of Tonic water is

in mixed drinks. Many avid drinkers enjoy a nice vodka or gin and tonic, the bitter sweet mix

allows for a delicious concoction especially garnished with lime or mint [3].

The use of Ultraviolet-Visible spectroscopy is to measure a solutions ability to absorb

different wavelengths of light. As described by the name this instrument specifically deals with
Ultraviolet and Visible wavelengths in the Electromagnetic spectrum. Specifically the UV-Vis

instrument used in this experiment was the Cary 100, which measured approximately between

145 nm to 800 nm wavelengths. As light passes through the sample it is absorbed by electrons

which causes the electrons to be excited and transition to higher energy levels. These

electronic transitions only occur when a certain amount of energy, or a specific wavelength,

passes through the molecule. The unabsorbed light passes through the sample hitting a

detector. This is then utilized to create a spectrum on a computer that shows which

wavelengths are absorbed by the molecule. Once the maximum wavelength is determined one

can take multiple solutions of the same molecule with unknown concentrations and create a

calibration curve. The reason this is possible is due to Beers Law, A=bc, where A is the

absorbance, is the molar absorptivity, b is the sample path length, and c is the concentration.

The absorbance is what is calculated from the computer. The molar absorptivity is the

corrected absorption value, this can either be calculated if the other variables are known or can

be looked up from previous texts. The path length is usually the width of the cuvette used for

sampling. Concentration is the moles of solute per liter of solution and can be found

algebraically or by dimensional analysis if the weight of solute and amount of solution is

known. Although Beers law will not be used in this lab it is still essential to the concept of UV-

Vis spectroscopy. For the purpose of this experiment, UV-Vis will be used to find the excitation

wavelengths of Quinine [4].

One purpose of Florescence Spectroscopy is to be able to identify the wavelength of

the photon that is emitted from molecules. After a molecule absorbs energy, an electron

transitions to higher electronic state. Once in the higher state it can drop to multiple
vibrational or rotational states or it can return to the ground electronic state. In the process of

transitioning from a high state to low state the electron emits energy in the form of a photon

of light, this process is known as luminescence. The main two forms of luminescence are

Florescence and Phosphorescence. Florescence is the faster of the two processes and will be

the one studied in this experiment. Used in tandem with UV-Vis, the excitation and emission

wavelengths of electrons in a molecule can be identified [5]. The Fluorescence

Spectrophotometer also contains a photomultiplier tube (PMT) where the user is able to

change the intensity detection limit of the sample in the instance of high and low readings.

This is used to determine the best amount of intensity that should be utilized in making the

calibration curve.

Electron transitions are an interesting topic to study. The use of UV-Vis and

Fluorescence allow scientists to see specific wavelengths of light absorbed and emitted by

electrons being transitioned. These are definitely important instruments for analytical

chemists due to the wide array of uses in the exploration of electron transition, vibrational, and

rotational states and forbidden transitions. It is applicable to many solution concentration

experiments; another way to create calibration curves based on two separate readings instead

of one, such as just using UV-Vis. Quinine fluoresces because of these transitions. At around

350 nm quinine transitions from S0 to S1, emitting light around 450 nm. At 450 nm quinine has

a transition from S1 back to S0. The S0 to S1 is the excitation and this relaxation of S1 back to S0

causes the fluorescence. [6]

This experiment will explore many important instrumental techniques, including

Ultraviolet-Visible (UV-Vis) Spectroscopy, Fluorescence Spectroscopy, and the creation and

use of calibration curves. The data from both types of spectroscopy were then used to

calculate the amount of Quinine in different brands of tonic water. The goals of this lab were to

learn how UV-Vis relates to Fluorescence, and how to utilize Fluorescence spectroscopy data

to determine concentration from absorbance, and how electrons have various transition states

that absorb and emit energy.

Procedure

A 1 L solution of 1 M H2SO4 was prepared from a concentrated 18 M H2SO4 obtained

from the stock room in order to prepare a stock solution of quinine. A ~100 ppm quinine stock

solution was prepared by adding 120.9 mg of quinine sulfate dihydrate from the stock room to

50 mL of 1 M H2SO4 and was diluted to 1 L with distilled water. Additionally, a 1 L solution of

0.05 M H2SO4 was prepared by diluting 3 M H2SO4 obtained from the stock room. Then a series

of quinine standards were prepared using a sequential 10x dilutions of quinine solutions

beginning with the 100 ppm stock solution. Five standards were prepared. The first standard

was prepared by taking 10 mL of the quinine stock solution and adding it to 40 mL of H2SO4

solution. The second standard was prepared by taking 10 mL of the first standard and adding it

to 40 mL of H2SO4. The other three standards were prepared the same way; all taking 10 mL

from the previous standard and adding it to 40 mL of H2SO4 solution.

The absorbance of the -100 ppm quinine stock solution was measured using the UV-vis

spectrophotometer in order to measure an absorbance spectrum, and the instrument was

zeroed with a 0.05 M H2SO4 solution.

 Excitation and emission spectrums were measured separately for all four standard

quinine solutions with the luminescence spectrometer. Based on the emission wavelengths

obtained from the emission spectra and the absorption wavelength determined by the UV-vis,

simple reads were performed at an excitation wavelength of 346 nm and an emission

wavelength of 450 nm. The intensity of the emitted light from the five quinine standards was

recorded. The unknowns tested were Giant Eagle brand tonic water and Fever Tree brand tonic

water. The group decided on testing the least expensive and most expensive tonic water

available in lab to test whether or not you are in fact getting higher quality tonic water when

you spend more money. The groups hypothesis stated that the Fever Tree tonic water would

contain a higher level of quinine than the Giant Eagle brand tonic water.

For this experiment, all the samples were scanned using variable intensities. This gave

the researchers an idea of the readings they should expect for the final scan. For the final

scans, all samples were set to the medium intensity preset, which proved to show the best

results for this experiment. These scans showed the optimal emission wavelength of the

molecule in question. The calibration curve was set up using the logarithm base ten of relative

fluorescence versus the logarithm base ten of the concentration of the samples [7]. Since no

specific concentration is stated one could use molarity or parts per million in the final analysis.

The data points one would use for this curve come from setting the wavelengths of excitation

and emission on the Fluorescent Spectrophotomer to the most optimal wavelengths found

from performing scans with the UV-Vis and Fluorescence.

Results

Before using the Varian Cary Eclipse Fluorescence Spectrophotometer, the excitation

and emission wavelengths needed to be found using the Cary 100 Bio UV-Visible

Spectrophotometer. Once the stock solution were run through the machine, there were two

peaks displayed for the absorption maximum- one at 346 nanometers and one at 450

nanometers. These wavelengths were then taken to the fluorescence spectrophotometer and

used to scan the samples. The second peak observed on the spectra is the emission

wavelength for quinine. As stated in the introduction, the quinine is excited around 350 nm and

the sample emitted light at 450 nm. The quinine stock solution and five diluted samples were

tested in the instrument.

First, they were run on the scanning mode so the settings of voltage and wavelength

could be best determined. During this time, the settings were adjusted to give the results with

the least noise. This included changing the PMT detector voltage value and adjusting the

excitation and emission spectra. The PMT detector adjusts the voltage and the group settled

on using a PMT level of Low for both the standard samples and unknowns. The low voltage

setting was used because intensity readings from the stock solution and the first diluted

standard were putting out readings above the instruments range. Once changed to Low, the

intensities were able to be picked up by the instrument. The intensity of the standards that

were able to be measured by the instrument are presented in Table 1, along with the

concentration and PMT detector settings used for each.

 After measuring the intensity of the standards the plotted data revealed a linear

relationship with the equation of y=1.0586x-0.4446 (Fig. 1). Once a calibration curve was made

the Giant Eagle tonic water and Fever Tree tonic water were measured in the simple reads. The

resulting intensities, concentrations and PMT detector voltages for the two unknowns can be

found in Table 2. The calculated concentration for the Giant Eagle Tonic Water was 60.5 ppm

and the concentration for the Fever Tree Tonic Water was 70.1 ppm.
 Table 1 displays the intensity readings, concentration and voltage (PMT) for two standards

used to create the calibration curve.

Fluorimeter Readings Excitation = 346 nm Emission = 450 nm

Sample Average Reading (Intensity) Concentration (ppm) PMT detector voltage

stock 94.88 100 L

A 9.866 10 L
 Table 2 displays the intensity readings, the calculated concentrations and the voltage (PVT) for

the two unknowns.

Fluorimeter Readings Excitation = 346 nm Emission = 450 nm

Sample Average Reading (Intensity) Concentration (ppm) PMT detector voltage

Giant Eagle Tonic Water 63.631 60.5 L

Fever Tree Tonic Water 73.707 70.08 L

Graph 1 displays the calibration curve using the intensity and concentration from Table 1.

Concentration of Quinine standards (ppm) vs.

Intensity of Quinine Standards (AU)
110
100
90
80
Intensity (AU)

70
60 y = 1.0586x - 0.4446
50 R = 1
40
30
20
10
0
0 10 20 30 40 50 60 70 80 90 100
Concentration (ppm)
Discussion

The goal of the experiment was to determine the concentration of quinine in different

brands of tonic water. Preparing standards with a quinine stock solution allowed for the

creation of a standard curve to then compare the intensities of the unknowns to the known

intensities of the standards. The only two standards plotted were the quinine stock solution

and one further dilution, known as dilution A. The other four dilutions (dilutions B, C, D, and

E) read negative for intensity meaning that the concentrations of quinine in those stock

solutions was too low to be read by the instrument. The unknown intensity readings fell

between the stock and dilution A readings so the negative data points were not included in the

graph. Another reason that the standard dilutions B, C, D and E read negative may be because

of quenching. Quenching can decreases the intensity of solutions through a variety of different

processes. This can cause fluorescence to disappear since intensity and concentration are

directly related. [8]

After removing the negative data points the graph displayed a linear progression with

an equation of y=1.0586x-0.4446. The y is the recorded intensity. The intensity of the Giant

Eagle tonic water was 63.631 AU. After plugging this value in for y the concentration of the

tonic water was shown to be 60.5 ppm. The intensity of the Fever Tree tonic water was 73.707

AU. After plugging this value in for y the concentration of the tonic water was shown to be

70.1 ppm. Fever Tree is a more expensive brand of tonic water than Giant Eagle and therefore

one might conclude that if you pay more money for it then it should be higher quality tonic

water. In the case of Giant Eagle verses Fever Tree this is true. Fever Tree tonic water has a

higher concentration of quinine than the Giant Eagle brand. The tonic water was measured
neat for the experiment. This was not optimum procedure- the article provided by the lab

instructor stated that the tonic water samples were to be diluted with the H2SO4 stock

solution. Because this was not done by the group, the calculations done did not have to

account for dilutions. If performed again, the group would make sure to dilute the tonic water

samples before measuring their intensity and comparing them with the standards.

Both fluorescence and UV-Vis were used in the experiment to determine the

concentration of quinine in tonic water. Both these methods are needed, but for different

purposes. First, in UV-vis, a range of wavelengths of light are passed through the sample, and

only certain wavelengths are absorbed by the sample being measured. The unabsorbed light,

as stated in the introduction is picked up by the detector and is used to make an absorption

spectra. In UV-vis there is a broad range of wavelengths, infrared and visible, that are used on

the sample. In fluorescence the wavelengths are picked individually for the light that gets

shined on the sample, excitation wavelength, and the light that comes off the sample,

emission wavelength. One added bonus to the fluorescence is the ability to change the voltage

in the instrument to adjust the intensity readings. Without this one would have to either

further dilute the standards and samples made or make them more highly concentrated. Using

the fluorescence spectrophotometer, the PMT detector voltage can be changed to Low if the

concentration of a sample is too high or it can be changed to High if the concentration is too

low. [6]

Sometimes excitation at different wavelengths gives the same emission spectra. This

can be explained through Kashas Rule and the Franck-Condon factor. When a molecule

absorbs the light from the UV-Vis, the electrons can be excited to many different higher
energy states. This is why we see different excitations for the same sample. According to

Kashas Rule, the emission is expected only from the lowest excited state, S1. [9] This is why

the same emission spectra is observed for different excitation wavelengths. The Franck-

Condon factor states that the greatest fluorescence will occur when S1 moves to the vibrational

state of S0. [6] The vibrational states above S1 can transition quickly to S1 and do not have time

to fluoresce. The gap between S1 and S0 is greater and thus when this transition takes place so

does fluorescence. [9]

Conclusion

When determining the concentrations of quinine within a matrix it is best to use both

UV-Visible spectrophotometers and fluorescence spectrophotometers. These methods work

together to produce an accurate representation of the amount of quinine within the standard

solutions and the unknown solutions. Using a standard curve made with the intensities

recorded by the fluorimeter and the known concentrations of the standards the group was able

to determine the concentration of quinine in the unknowns. Using the linear trend line

produced from Graph 1 the concentration of quinine in Giant Eagle tonic water was 60.5 ppm

and the concentration of quinine in Fever Tree tonic water was 70.1 ppm. This experiment

provided evidence that higher quality tonic water does in fact contain higher concentrations of

quinine than lower quality tonic water.

 Bibliography

1. "Quinine: Indications, Side Effects, Warnings." Drugs.com. Drugs.com, 01 Feb. 2017. Web.

13 Feb. 2017.

2. "Quinine." U.S. National Library of Medicine. National Institutes of Health, 06 Dec. 2016.

Web. 13 Feb. 2017.

3. Anthony, Kyle B. "5 Benefits of Tonic Water." Made Man. Made Man, 06 July 2010. Web. 13

Feb. 2017.

4. Reusch, William. "UV-Visible Spectroscopy." UV-Visible Spectroscopy. IOCD, 05 May 2013.

Web. 13 Feb. 2017.

5. O'Reilly, James E. "Fluorescence Experiments with Quinine." Journal Of Chemical Education

52.9 (1975): 610-12. Web. 13 Feb. 2017.

6. Granger, R., Yochum, H., Granger, J., & Sienerth, K. (2017). Atomic Absorption

Spectroscopy. In R. Granger, H. Yochum, J. Granger, & K. Sienerth, Instramental

Analysis (pp. 207-242). New York: Oxford University Press.

7. "Introduction to Fluorescence." Principles of Fluorescence Spectroscopy (2006): 1-26.

PerkinElmer, 2000. Web. 13 Feb. 2017.

8. Clark, W. (2017, February 14). Reasons for Negative Standard Solution Readings . (M.

McCarty, Interviewer)

9. Louis, G., & Kumar, S. (2008). Unusual autofluorescence characteristic of cultured red-rain

cells. SPIE, 1-9.