EXPERIMENTAL ANALYTICAL CHEMISTRY

II
LW1. Molecular Spectrophotometric methods
(UV-Vis and Fluorescence)

September 30 2021
Aims of these lab works
In order to achieve the aims stated for this Course, the following partial objectives
are pursued in these lab works:

• To become initiated in calibration methodology for the instrumental methods

of analysis using the External calibration method.

• To experiment with analytical procedures based on spectrophotometric

measurements.

• To understand the methodology of instrumentally-based analytical procedures.

• To become initiated in reporting tasks (lab notebook and short or long report).

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1 LW1a: SPECTROPHOTOMETRIC
DETERMINATION OF TOTAL IRON IN
WHITE WINE

1.1 Theoretical background

1.1.1 Spectrophotometry

Spectrophotometric measurements are based on the determination of the amount of energy that,
at a given wavelength in the Ultraviolet-Visible spectrum, a dissolved substance absorbs from the
total energy beamed from an external radiation source. These measurements are taken with a
spectrophotometer, whose basic parts are the following:
• In the first place, the light source. This is normally a wolfram filament whose emitted beam
is directed through a monochromatic filter to be able to work at a selected wavelength from
the UV-Vis spectrum.
• The monochrome light beam is made to cross a cell where the solution containing the analyte
to be determined is placed. This light beam travels a fixed and known length (which is also
called the ’optical path’) and is partially absorbed and the rest is transmitted.
• The transmitted part arrives at the detector and produces an electrical signal that is amplified
and converted to digital form. Depending on the spectrophotometer’s electronics, the user
can obtain this information as %T (percent transmitted light) or Absorbance (amount of
absorbed light) of the sample.

Figure 1: Scheme of a model spectrophotometer.

The absorbance (A) is linearly correlated to the analyte concentration in the solution (c) through
Beer-Lambert’s law:

Aλ = ebc

where: Aλ = Absorbance at a given wavelength; e = molar absorptivity; b =optical path (cm); c

= molar concentration

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1.1.2 Spectrophotometric determination of Fe in white wine

In this lab work, the total amount of iron in a white wine is to be determined. The white wine is
made using white or red grapes (with the latter, the skin is immediately eliminated to prevent
coloring the wine). During this process, the acidity and the compounds that are present in
wine vary depending on the grape type, the process’ characteristics and the material used.
The iron present in wine should be due to the original grape iron content and the metallic
material used during the process. The total iron present in the wine can be determined using
spectrophotometric measurements based on the chemical reaction between Fe(II) and 1,10-
phenantroline according to the following reaction:

It is compulsory that all the iron species present in the samples be in the form of Fe(II) to determine
the total amount of iron, so it is necessary to reduce the possible Fe(III) content to Fe(II). This can
be done by using hidroxylamine chlorhydrate as a reducing agent, according to the following
chemical reaction:
4 Fe3+ + 2 NH2OH ⇐⇒ 4 Fe2+ + N2O + 4 H+ + H2O
The reaction for the complex formation needs a pH in the 2 - 9 interval, in other words, wide
enough to cope with the amount of acid released in the last reaction. The complex formed,
consisting of Fe(II) and 1,10-phenantroline has a red color, and its maximum absorbance is
observed at a wavelength that must be studied experimentally in this work. In that wavelength, the
color intensity measured follows Beer-Lambert’s law, which can be solved for the Fe (II)
concentration according to:

𝐴𝜆𝑚𝑎𝑥
𝐶𝐹𝑒 ,𝑡𝑜𝑡 =
𝜀𝑏

The value of the optical path (b) is 1 cm and the molar absorptivity (e) is calculated experimentally
in the calibration process. This value is the slope of the calibration curve, which must be
constructed before measuring the Fe concentration in the white wine sample.

1.2 Operating procedure

1.2.1 Reagents and solutions (for each couple)

• Prepare the needed volume of iron stock solution, approx. 2 10−4 M, from hexahydrated
iron ammonia sulfate (MW 392.14 g/mol) by weighing the adequate amount of this reagent.
This Fe(II) solution will be used to prepare the standard solutions for the method calibration.
Calculate the Fe(II) exact molar concentration in this solution!
• Prepare the needed volume of approx. 2 10−3 M solution of 1,10-phenantroline (MW
198.23g/mol). Before add water, dissolve 1,10-phenantroline in 5mL of methanol.
• Prepare the needed volume of approx. 10% w/v solution of hydroxylamine chlorhydrate
(MW 69.49 g/mol).
• Prepare the needed volume of approx. 0.1 M solution of sodium acetate.
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1.2.2 Preliminary Experiments
In order to investigate the wavelength in which the maximum absorbance for the Fe(II) / 1,10-
phenantroline complex is observed (λmax ) and to test its formation kinetics, it is interesting to
carry out a small experiment in which the spectra of this complex is measured at fixed time
intervals.
To this end, prepare 50 mL of a solution containing 10 mL of the working Fe(II) stock solution,
along with 2 mL of Hydroxylamine chlorhydrate solution, 10 mL of sodium acetate solution
and 5 mL of the 1,10-phenantroline solution. Finally, add water up to the mark. As soon as
you have prepared this, put it in the sample holder of the Diode-Array Spectrophotometer and
begin recording spectra at 1-min time intervals for at least 15 min1. Examination of this
collection of spectra should give you a hint about both λmax and the time needed for the
formation of the absorbing iron complex.

1.2.3 Sample preparation

Each student pair shall measure one wine sample, which will be done in triplicate in order to have
enough information to apply more advanced statistical procedures to check the total iron amount
in the wine samples.
According to the type of sample (wine), it may be advisable to filter it before proceeding.

1.2.4 Analytical method calibration and unknown samples measurement

• Preparation of the standard solutions

To obtain the calibration curve, first clean six 50 mL volumetric flasks. The volume of the
working Fe(II) stock solution needed to prepare a calibration set of solutions (consisting of
5 concentration levels and the blank) that adequately covers the expected Fe
concentration range in the wine sample should be calculated previously. Then, add 2
mL of Hydroxylamine chlorhydrate solution, 10 mL of sodium acetate solution and 5 mL
of 1,10-phenantroline solution to each of the 6 flasks and fill with distilled water to the
mark. Stir to homogenize the solution and wait for 10 minutes before measuring.
• Construction of the calibration curve
Once the spectrophotometer is on (let it warm up at least 15 min) and set to the λMax nm
wavelength, fill the spectrophotometric cell with distilled water and set the instrument to
zero absorbance. This should be always done with the same cell used for the calibration and
for the measurement of the unknown samples. Transfer a portion of each calibration solution
to the cell. Pour the cell’s content and fill it again with the same solution. Dry the cell’s
walls and insert it into the spectrophotometer to take the absorbance measurement at λMax
nm.
Each measurement should be made in triplicate. Once the 6 calibration solutions have been
measured, 6x3 data pairs (Aλmax , [Fe(II)]) will be available. With these data, the calibration
curve (or regression line) can be calculated according to the criteria explained in class. When
the calculations are completed, and once it is clear that there is no need to repeat any of
the measurements, the calibration process of the analytical method is concluded.
• Measurement of the wine sample
Take 25 mL of wine sample and add it to a 50 mL volumetric flask. Then, add 2 mL of

1 1
Your instructors will give you indications so as to adequately arrange the instrument for these
measurements

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 hydroxylamine chlorhydrate solution, 10 mL of sodium acetate solution and 5 mL of 1,10-
phenantroline solution to each flask and fill with distilled water to the mark. Stir to
homogenize the solution and wait for 10 minutes before measuring. Flush the
spectrophotometric cell, empty it and re-fill it again with the sample solution. Measure the
absorbance at λMax nm in triplicate. With this information, calculate the total iron amount
in the wine and its uncertainty using the calibration information.
• Additional work
In the event the lab work was completed satisfactorily, the preparation of certain extra
standard solutions to complete quality requirements of the analytical procedure can be
considered. One of these extra solutions is the preparation of 3 blank samples to
estimate the limit of detection. Another set of extra solutions would be preparing
standards based on the addition of 1, 5 and 9 ml of the working Fe(II) stock solution and
to consider them as an external set of solutions to estimate the accuracy and precision of
the calibration curve. Details on these two tasks are given by the lecturers.

1.3 Results and reports

In general terms, there are certain tasks that should be carried out and skills that should be
taught before entering the lab. These include calculations for the preparation of standard
solutions, the calculation of the recoveries and how the final results are obtained from the
experimental measurements. Over the course of experimental work, it is rather difficult to
efficiently combine these required skills and all the duties of the lab work. This circumstance will
be closely scrutinized by the lecturers in charge of lab work.
Firstly, the laboratory notebook is where you must complete the scheme of the work before
entering the lab, where you must make note of all the experimental data and observations, as well
as the intermediate and final results. This notebook will also be evaluated.
Based on the data collected, your reports should contain information about the following topics
(if applicable):

• Calibration
螈٦H Range of calibration (the most reliable regression line)
螈٦H Uncertainty calculation of the concentration obtained by the regression line
• Analysis
螈٦H Final results (average and standard deviation, taking into account the whole procedure)
螈٦H 螈٦卐٨Che analyte’s final concentration at 95% of confidence
螈٦H Comparison of results (among couples and/or real value)

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2 LW1b: FLUOROMETRIC DETERMINATION OF
QUININE IN TONIC WATER

2.1 Theoretical background

2.1.1 Fluorescence spectrophotometry

Luminescence involves emission of photons from excited atoms or molecules. Fluorescence and
Phosphorescence, both luminescent processes, involve emission of photons from systems that
have been excited by the absorption of photons. In molecular Fluorescence Spectroscopy, the
analyte molecule first absorbs a photon (excitation, ∆Eexcit) that leaves the analyte in an
electronically and vibrationally excited state.
At this point, the molecule rapidly loses excess vibrational energy by non-radiatively relaxing to the
ground vibrational level of the excited electronic state. This occurs because energy is transferred to
solvent molecules as the analyte collides against them. This relaxation process is very efficient and
very rapid. Finally, the molecule can fluoresce (∆Erelax). This process is represented schematically
in Figure 2.1.
The fluorescent intensity is proportional to the radiant power absorbed by the sample, and taking
Beer-Lambert’s Law into account, which states that absorbed intensity is proportional to sample
concentration

𝐴𝜆 = 𝜀𝑏𝑐

it could be concluded that the fluorescent intensity is proportional to the concentration of the
analyte:

𝑖𝐹 = 𝑘𝑐

where: iF = fluorescent Intensity, k = constant and c = molar concentration.

Figure 1: The fluorescence process.

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The fluorometric methods are very sensitive and selective. Because of the high sensitivity of
the approach, it is important to work carefully and with clean glassware to avoid
contamination.

Quinine (see Figure 2.2), originally derived from the bark of the Cinchona tree, is an effective cure
for malaria. Traditionally, because of its bitter taste, Quinine was mixed with gin, giving rise to
the Gin and Tonic cocktail. Quinine is used as a flavoring agent in Tonic Water, Bitter Lemon and
Vermouth. Its concentration is limited by the US Food and Drug Administration to 83 mg/L;
with most commercial preparations at the 25 – 60 mg/L level. In this lab work, the amount
present in a commercial product will be measured.

Figure 2: Molecular structure of quinine.

2.1.2 Fluorometric determination of quinine in tonic water

In this lab work, the amount of quinine in tonic water is to be determined. The analysis of
quinine in tonic water is simplified by taking advantage of the high sensitivity and selectivity of the
method used. Quinine is a very efficient fluorescer, with an excitation wavelength at 350 nm1.
Emission occurs at 450 nm. Throughout this lab work, it is important to remember to set up the
spectrofluorometer at the ’Low’ sensitivity scale in order to obtain an adequate calibration
range2. The tonic water matrix can be considered relatively complex, but any matrix effect is
essentially eliminated by dilution of the tonic water in an acidic aqueous solution. This can be
done because, even after significant dilution, the quinine remains at a detectable
concentration. The selectivity of the approach also means that other components in the tonic
water do not generate a signal, and are thus not detected.

2.2 Operating procedure

2.2.1 Reagents and solutions (for each couple)

• Prepare the needed volume of approx. 0.5 M H2SO4 solution for each group.
• Prepare the needed volume of quinine stock solution at 100 mg/L concentration in 0.5 M
H2SO4. It is important to record the actual concentration of this stock solution in the Lab
Notebook. This quinine solution will be used to prepare the standard calibration solutions
for the method calibration. Calculate the quinine’s exact molar concentration in his
solution!. Quinine is in the form of sulfate dehydrate 2C 20H24N2O2.H2SO4.2H2O (MW
782.96 g/mol)

1250 nm can also be used.

2Setting it to ‘High’ (the instrument’s default state) will force the use of at least a 5-fold dilution of the calibration
standards in order to avoid instrumental signal saturation.

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2.2.2 Tonic water preparation

• Each pair of students must measure one tonic water sample in triplicate in order to obtain
enough information to apply more advanced statistical procedures to verify the total quinine
amount in tonic water.
• Carefully clean and rinse a 25 mL volumetric flask.
• Pipette 0.25 mL of degasified tonic water3 into the 25 mL volumetric flask and dilute with
0.5 M H2SO4. Prepare three samples to analyze precision.

2.2.3 Analytical method calibration and unknown sample measurements

• Preparation of the standard solutions

To obtain the calibration curve, first clean six 25 mL volumetric flasks. Prepare six standards
of approx. 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/L by diluting the stock standard solution. Start
with the lowest concentration standard. Use the appropriate micropipette to introduce the
adequate calculated volume of the stock solution into the volumetric flask and dilute to 25.0
mL with 0.5 M H2SO4.
Calculate the actual concentrations of these standards and record the values in the Lab
Notebook.
• Construction of the calibration curve
Once the spectrophotometer is on (let it warm up at least 15 min) and set to the excitation
(250 or 350 nm) and emission wavelength (450 nm), fill the fluorophotometric cell with
distilled water and set the instrument to zero absorbance. This should be always done with
the same cell used for the calibration and for measurement of the unknown samples.
Transfer a portion of each calibration solution to the cell. Pour the cell’s content and fill it
again with the same solution. Dry the cell’s walls and insert it in the spectrofluorometer
to take the absorbance measurement at 450 nm. Each measurement should be taken in
triplicate.
Once the 6 calibration solutions have been measured, 6x3 data pairs (emission, [quinine])
will be available. With these data ,the calibration curve (or regression line) can be
calculated according to the criteria explained in class. When the calculations are
completed and once it is clear that there is no need to repeat any of the measurements, the
calibration process of the analytical method is concluded.
• Measurement of the tonic water sample
Flush the fluorophotometric cell, empty it and refill it with the sample solution again.
Measure the absorbance at 450 nm in triplicate. With this information, calculate the total
quinine amount in the tonic water and its uncertainty using the calibration information.
• Additional work
In the event the lab work was completed satisfactorily, the preparation of certain extra
standard solutions to complete quality requirements of the analytical procedure can be
considered. One of these extra solutions is the preparation of 3 blank samples to
estimate the limit of detection. Another set of extra solutions would be preparing
standards of 0.1, 0.5 and 0.9 mg/L following the aforementioned procedure, and to
consider them as an external set of solutions to estimate the accuracy and precision of the
calibration curve. Details on these two tasks are given by the lecturers.
3This process can be carried out in the ultrasonic bath, but you must take great care. It is wise to open the
bottle in advance and to transfer the content from one beaker to another in order to remove much of the gas
before using the ultrasonic bath.

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2.3 Results and reports

In general terms, there are certain tasks that should be carried out and skills that should be
taught before entering the lab. These include calculations for the preparation of standard
solutions, the calculation of the recoveries and how the final results are obtained from the
experimental measurements. Over the course of experimental work, it is rather difficult to
efficiently combine these required skills and all the duties of the lab work. This circumstance will
be closely scrutinized by the lecturers in charge of lab work.
Firstly, the laboratory notebook is where you must complete the scheme of the work before
entering the lab, where you must make note of all the experimental data and observations, as well
as the intermediate and final results. This notebook will also be evaluated.

Based on the data collected, your reports should contain information about the following topics
(if applicable):

• Calibration
螈٦H Range of calibration (the most reliable regression line)
螈٦H Uncertainty calculation of the concentration obtained by the regression line
• Analysis
螈٦H Final results (average and standard deviation, taking into account the whole procedure)
螈٦H 螈٦卐٨Che analyte’s final concentration at 95% of confidence
螈٦H Comparison of results (among couples and/or real value)

10