# Calculate the Molarity of the solution

Please calculate the molarity as asked in the pic attached, showed calculations

PHU22101 Lab Report
Experiment 2 – HPLC-Fluorescence
Student Name: Caitriona Horgan
Group Number: C
Date: 22/02/2021
Demonstrator:
1. Introduction to the theory underlying this experiment
(background to the principles of the technique, with special reference to the experiments
performed, chemical structures & reactions, expected outcome of the experiment)
→ do not write more than one page in this section
2. Results and data analysis
1. Standard Curve (in person)
How were the dilutions for the standards carried out?
Concentration
Peak Height
Peak Area
0.1 ppm
5197
51651
0.5 ppm
35924
350647
1 ppm
90848
888856
2 ppm
202665
2001276
3 ppm
301715
2976011
0.01 ppm
Which of the graphs will be used as a basis for the following quantifications and why?
It would be more suitable to use the graph of peak height against concentration as this graph has R2
value closer to 1, indicating the correlation between peak height and concentration is stronger than
the correlation between peak area and concentration.
2. Tonic Water Samples (in person)
Data obtained for tonic water samples:
Tonic Water Brand
Peak Height
Peak Area
Dilution
Tonic Scheppes
124150
1239732
10
Tonic Club
136962
1370084
10
Calculation of Quinine content in Tonic Water samples:
Tonic Scheppes:
y = 75978x – 100663
124150 = 75978x – 100663
x = 2.9589 ppm
(2.9589)(10) = 29.589 ppm
Club Tonic Water:
y = 75978x – 100663
136962 = 75978x – 100663
x = 3.1276 ppm
(3.1276)(10) = 31.276 ppm
3. Actavis® Quinine Sulfate Tablets (in person)
How were the dilutions for this experiment carried out?
1) Weight of equivalent of 10 mg quinine sulfate in tablet [mg]
Weight of tablet = 688 mg
We know there is 300 mg in 688 mg
We want 10 mg in x mg
Cross multiply, 6,880 mg in 300x
Therefore, 22.9333 mg is the weight equivalent we need.
2) Concentration of measured solution in mg/100 ml
3) Concentration of measured solution in ppm
Complete the table below
Weight of Tablet [mg]
688 mg
Weight of equivalent of 10 mg quinine sulfate in tablet [mg]
23 mg
Concentration of measured solution in mg/100 ml:
Concentration of measured solution in ppm:
Peak height
67303
Peak area
688802
4) Calculation of Quinine content in tablets
y= 75978x – 100663
67303 = 734972x – 21601
x = 0.12096 ppm
BP monograph states that the content of quinine sulphate should be 95-105% of the stated
amount.
(Concentration calculated from graph/concentration of measured solution) x 100
(0.12096/1) x 100 = 12.096 %
Therefore, the sample does not comply. The sample may not comply due to an error in the
manufacturing of the tablet, inaccuracies in weighing crushed tablet or diluting the solution. The
solution may also not have had an even distribution of solute.
4. pH Dependence (online)
pH
(theoretical)
pH
(measured)
Relative
Fluorescence
Intensity
1
1.10
2.8
2
1.87
6.9
3
2.96
55.4
4
3.86
45.6
5
4.88
22.3
6
5.93
6.4
The results obtained indicate that the optimum pH was at pH 2.96. Upon further reading following
the experiment, it was discovered that the optimal pH for the fluorescence of quinine sulphate is
approximately 4.1. Quinine sulphate is known to strongly fluoresce in a dilute acidic solution.
5. Halide Quenching (online)
Complete the table below – calculate the molarity of the NaBr solutions (show calculation).
ml of
0.05 M NaBr
Molarity of
NaBr [mM]
Relative
Fluorescence
Intensity
1
17,649
2
13,788
4
11,060
8
6,695
16
5,100
Calculations:
Plot a graph with NaBr concentration vs. relative fluorescence intensity and comment on the graph
observed.
3. Questions
1. Identify functional groups in quinine, name the heterocycles,
assign pKa values (pKa1 = 4.1, pKa2 = 8.5), and identify stereocentres.
2. What electrodes are used for pH measurements?
3. Why does increasing concentration of bromide reduce relative fluorescence intensity?
Increasing bromide concentration results in a reduction in relative fluorescence intensity due to
collisional quenching. Collisional quenching occurs when the excited chromophore clashes with
an atom or molecule that can facilitate non-radiative transitions to the ground state without the
emission of a photon. The bromide ion acts as the quenching agent in this experiment. The
increase in bromide concentration results in an increase in collisions which in turn causes a
greater reduction of relative fluorescence activity.
4. Discuss other quenching processes which compete with fluorescence.
Intersystem crossing is a deactivation process that competes with fluorescence. In this process,
the electron in the equilibrium singlet state can change its spin resulting in the formation of a
triplet. Energy can be emitted as light. Resonance energy transfer is another deactivation
process whereby energy can be transferred without direct interaction to a second, acceptor
molecule. It decreases the energy of a donor and transfers the energy to an acceptor. Other
deactivation processes include quenching (which has already been discussed) and vibrational
energy loss.
5. Explain why emitted radiation in fluorimetry is measured at 90° from the excitation light.
Fluorescence is measured at 90° from the excitation radiation to prevent incident light from
interacting with the detected fluorescent light, which would lead to inaccurate values being
obtained. Measuring at 90° also makes it possible to measure samples from the front when they
are opaque or turbid by measuring the emitted light.
6. Describe and illustrate the ‘experiment’ carried out by Stokes which led to the discovery of
fluorescence.
Evaluation of the practical procedure – did anything not go according to plan? Do you have any
suggestions to improve this practical?
What do you think you have learnt in this lab/when writing up this lab report?

Plagiarism statement:
Title: HPLC – Fluorescence
Scope:
ANALYSIS 02
1. Determination of Quinine in Soft Drinks using Fluorescence Detection
2. Determination of Quinine in Actavis® Quinine Sulfate Tablets
(300 mg) by HPLC Using a Spectrofluorimeter
3. Quenching by pH and halides
References: Samanidou, VF et al. Simple and Rapid HPLC Method for the
Determination of Quinine in Soft Drinks Using Fluorescence Detection.
J. Liquid Chromatogr. Rel. Technol., 2004, 27(15), 2397 – 2406.
BP Vol. III: Quinine Sulfate Tablets
Quinine (6′-methoxycinchonan-9-ol), a natural occurring alkaloid, is a bitter tasting
powder extracted from the bark of the cinchona tree of South America. It is well known
for its activity against malaria and, for this reason, has been used in medicine as antimalarial agent (dose of approx. 2000 mg/day for malaria prevention). Moreover, it has
been prescribed for the treatment of muscle cramps. Due to its bitter taste, it is also
used as an additive in soft drinks, such as tonic water and bitter lemon. However,
quinine is a potentially toxic drug. The typical syndrome of quinine side effects is called
cinchonism, and it can be mild in usual therapeutic dosage or severe in larger doses.
Quinine side effects include ringing in the ears, vertigo, disturbed vision, nausea,
diarrhoea, abdominal pain, headache and fever, renal failure, chest pain, and asthma.
For these reasons it should not be prescribed during pregnancy, as it can cause birth
defects and miscarriages. As a consequence, some countries, such as the United
States and Germany, order that quinine concentration must be declared on food labels
(with an upper limit of 83 ppm (mg/kg). 71 ppm of quinine are present in Schweppes
Tonic Water. In other countries such as China, quinine is not legally permitted to be
Quinine is a strongly fluorescent compound in dilute acidic solution with two excitation
wavelengths (250 and 350 nm) and a fluorescence emission at 450 nm. Factors
reducing the fluorescence emission (such as collisional quenching and chemical
quenching) will be studied as well as the use of emission spectra for quantitation.
Quinine has two pka values, 8.5 and 4.1.
BP Monograph – Quinine Sulfate Tablets:
MW 783 g/mol
Content of quinine sulfate, (C20H24N2O2)2, H2SO4, 2H2O
95.0 to 105.0% of the stated amount.
Storage: Quinine Sulfate Tablets that are not sugar-coated should be protected from
light.
300 mg of Quinine Sulfate is approximately equivalent to 248.6 mg of anhydrous
quinine.
Procedure: Read fully before starting analysis
———————————————————————————————————–HPLC
Instrument conditions:
Column:
Symmetry C18
Mobile phase:
Acetonitrile / methanol / 0.1 M Ammonium acetate 45:15:40%
Detector:
350 nm (excitation), 450 nm (emission)
gain: 1
EUFS: 10000
1 ml/min
Flow rate:
———————————————————————————————————–Standards (in-person)
1.0
Prepare a series of quinine sulfate solutions from a 100 ppm (= 100 µg/ml)
stock solution: 0.1 ppm, 0.5 ppm, 1 ppm, 2 ppm and 3 ppm with 0.05M H2SO4.
1.1
Transfer the standard solutions into a 1 ml injection vials, label and cap.
1.2
Place all of your samples into a beaker labelled with your name and group.
The HPLC will run over-night. You will receive an email with your data in the following
days.
1.3
Record Peak Height and Peak Area in your Lab Report.
———————————————————————————————————–Tonic Water Samples (in-person)
2.0
An indicative concentration of up to 20 ppm of quinine is given for tonic water
samples.
2.1
After opening the soft drink container, transfer an aliquot into a glass beaker,
and sonicate for 10 min in an ultrasonic water bath to remove carbon dioxide.
2.2
Perform sample dilution with 0.05M H2SO4 to reach the working range.
2.3
Transfer samples into 1 ml injection vials, label and cap.
2.4
Place all of your samples into a beaker labelled with your name and group.
2.5
Record Peak Height and Peak Area in your Lab Report.
Final Report: Determine the amount of Quinine Sulfate in the different Tonic Water
brands.
Actavis® Quinine Sulfate Tablets (300 mg) (in-person)
3.0
Weigh one quinine sulfate tablet, record weight in your Report Sheet and
powder the tablet.
3.1
Weigh a portion of the powder equivalent to 10 mg quinine sulfate. Record
3.2
Transfer powder to a 100 ml volumetric flask and add 80 ml of methanol.
Sonicate for 10 min in an ultrasound water bath and dilute to volume with
methanol.
3.3
Filter the solution – using a 0.45 um filter and a 2 ml syringe (repeat twice, to
a total of 6 ml) into a beaker.
3.4
Using a pipette, take 1 ml of this solution and transfer to another 100 ml
volumetric flask and dilute to volume with the HPLC mobile phase.
3.5
Transfer into a 1 ml injection vial, label and cap.
3.6
Place all of your standards and samples into a beaker labelled with your name
and group.
3.7
The HPLC will be run over-night. You will receive your results in the following
days by email.
Final Report: Determine the amount of Quinine Sulfate in one tablet (remember
dilutions!).
4.1
Plot 1) peak height (relative fluorescence intensity) vs. quinine concentration and
2) peak area vs. quinine concentration from the standards. Decide whether peak
height or area are more suitable for the following quantitative calculations.
4.2
Determine the concentration of unknown quinine samples (Tonic water), and
using a least squares spreadsheet, report as ppm in original sample.
4.3
Determine whether the quinine sulfate tablets comply with the limits given in the
Pharmacopoeia.
————————————————————————————————————
pH Dependence (ONLINE only):
5.0
Dilute the quinine stock solution (100 µg/ml) 1:10 by taking 5 ml and transferring
them to a 50 ml volumetric flask. Dilute to volume with 0.05 M H2SO4. (You will
need this solution for the next experiment (6.0 Halide Quenching) as well.)
5.1
Pipette 2 ml of this solution into six 25 ml volumetric flasks each and dilute to
volume with buffers pH 1, 2, 3, 4, 5 and 6.
5.2
Measure and record the exact pH of each solution (all containing equal amounts
of quinine).
5.3
Measure the relative fluorescence intensity for each solution (Ex 350 nm, Em
450 nm), using the respective buffer solution as a blank:
Instrument: RF-6000 Spectrophotometer
5.3.1 Set Excitation wavelength by selecting EX 350 nm and press enter.
5.3.2 Set Excitation wavelength by selecting EM 450 nm and press enter.
5.3.3 Gently open sample compartment
5.3.4 Place a solution of your blank* into a clean and dry fluorimetry cuvette.
(*specific pH or 0.05 M H2SO4.)
5.3.5 Making sure that all sides are clean place the cuvette into the cell holder
and close the compartment.
5.3.6 Select Auto Zero.
5.3.7 Place a solution of your sample into a clean and dry fluorimetry cuvette.
5.3.8 Making sure that all sides are clean place the cuvette into the cell holder
and close the compartment.
5.3.9 After 30 seconds press start. The reading will be recorded on the screen
table. Record fluorescence intensity on your Report Sheet.
5.3.10 Repeat 5.3.4 to 5.3.9 for each sample (with new blanks each time).
5.4
Determine the optimum pH for fluorescence detection of quinine and record in
Final Report: Plot a graph with pH vs. relative fluorescence intensity and comment on
the graph observed.
————————————————————————————————————
Halide Quenching (ONLINE only):
6.0 Pipette 2 ml of the 10 µg/ml solution, prepared under 5.0 pH Dependence above,
into five 25 ml volumetric flasks each, add 1, 2, 4, 8 and 16 ml of 0.05 M NaBr
solution and dilute to volume with 0.05 M H2SO4.
6.1 Calculate the molarity of NaBr of each of the solutions and record in your Report
Sheet.
6.2 Measure the relative fluorescence intensity for each solution (Ex 350 nm, Em 450
nm). Blank the instrument before each measurement with with 0.05 M H2SO4. See
5.3 in previous section for an SOP.
6.3 Record fluorescence intensity in your Report Sheet.
Final Report: Plot a graph with concentration NaBr vs. relative fluorescence intensity
and comment on the graph observed.
————————————————————————————————————
Questions:
1. Identify functional groups in quinine, name the heterocycles, assign pKa values
(pKa1 = 4.1, pKa2 = 8.5), and identify stereocentres.
2. What electrodes are used for pH measurements?
3. Why does increasing concentration of bromide reduce relative fluorescence
intensity?
4. Discuss other quenching processes which compete with fluorescence.
5. Explain why emitted radiation in fluorimetry is measured at 90° from the excitation
light.
6. Describe and illustrate the ‘experiment’ carried out by Stokes which led to the
discovery of fluorescence.
Complete the table below – calculate the molarity of the NaBr solutions (show calculation).
ml of
0.05 M NaBr
Molarity of
NaBr [mm]
Relative
Fluorescence
Intensity
17,649
1
13,788
N

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