EXPERIMENTAL ANALYTICAL

CHEMISTRY
2021-2022 LAB WORK 1
Two different works:

• Spectrophotometric Determination of Total Iron in White Wine

• Fluorometric Determination of Quinine in Tonic Water

Aims of the work:
1. To get initiated in the calibration methodology:
• External calibration method
2. To experiment with analytical procedures based on
spectrophotometric measurements
3. To experiment the applicability of analytical procedures in the field
of quality control
 LW 1a: Spectrophotometric Determination of Total Iron in White
Wine

Spectrophotometry:
• Based on determination of amount of UV-Vis spectrum wavelength
energy is absorbed by a dissolved substance.
• The absorbance (A) is linearly correlated to the analyte concentration
in the solution:

As ,  bc

Beer-Lambert ´s law
 LW 1a: Spectrophotometric Determination of Total Iron in White
Wine

Spectrophotometer:
 LW 1a: Spectrophotometric Determination of Total Iron in White
Wine

Spectrophotometric determination of Fe in white wine:

• Sources of iron:
o Grape iron content
o Metallic material used during process
• Fe(II) and 1,10-fenantroline complex formation
reaction takes place in a pH 2-9 range
• The maximum absorbance of the complex is at 510 nm
 LW 1a: Spectrophotometric Determination of Total Iron in White
Wine

Spectrophotometric determination of Fe in white wine:

• Taking in account Beer-Lambert´s law:

Fe(III) experimentally
Fe TOT 
A510
Fe(II)
b calibration

• Calibration process:
• External
 LW 1a: Spectrophotometric Determination of Total Iron in White
Wine

Reagents and solutions:

• ?? mL, 2·10-4 M Fe(II) stock solution (couple)
o Calculate exact concentration to use it as standard solution (from
hexahydrated iron ammonia sulphate )
• ?? mL, 2·10-3 M 1,10-fenantroline (couple)
• ?? mL, %10 hydroxilamine chlorhydrate (couple)
• ?? mL, 0.1 M sodium acetate (couple)
 LW 1a: Spectrophotometric Determination of Total Iron in White
Wine
Preliminary experiments:
• Determination of the maximum absorbance:
o 50 mL solution:
o 10 mL Fe(II) solution
o 2 mL hydroxylamine clorhydrate
o 10 mL sodium acetate
o 5 mL 1,10-fenantroline
o Diode-array spectrophotometer
o Record spectra at 1 min for 15 min (study of the kinetics)
 LW 1a: Spectrophotometric Determination of Total Iron in White
Wine

External calibration:
• Calibration solutions:
o Fe(II) stock solution (0, 2, 4, 6, 8 and 10 mL)
o 2 mL of hydroxylamine chlorhydrate solution
o 10 mL sodium acetate solution
o 5 mL of 1,10-fenantroline solution
o Wait 10 minutes before measuring
 LW 1a: Spectrophotometric Determination of Total Iron in White
Wine

External calibration:
• Calibration curve construction at maximum absorbance wavelength:
o Clean and homogenize the sample cell
o Make absorbance measurements (3 replica for solution)
o Build calibration curve
- Statistical treatment
 LW 1a: Spectrophotometric Determination of Total Iron in White
Wine

External calibration:
• Measurement of the wine sample
o 25 mL of wine, 50 mL volumetric flask
+ 2 mL of hydroxylamine chlorhydrate + 10 mL of sodium
acetate + 5 mL of 1,10-fenantroline
o Wait 10 minutes before measuring (triplicate) at selected
wavelength
o Calculate the total iron amount
 LW 1a: Spectrophotometric Determination of Total Iron in White
Wine

Results and reports:

• Calibration:
o External calibration (the most reliable regression line)
o Uncertainty calculation of the concentration obtained by the
regression line
• Sample concentration:
o The analyte’s final concentration at 95% of confidence
o Comparison of results (among couples and/or real value)
 LW 1b: Fluorometric Determination of Quinine in Tonic Water

Fluorescence spectrophotometry:
• Fluorescence involve emission of photons from previously excited
substances:
• Fluorescence intensity is
proportional to the concentration:

F  kc
 LW 1b: Fluorometric Determination of Quinine in Tonic Water

Quinine:
• It is an effective cure for malaria and is used as a flavouring agent
• Excitation wavelengths: 250 and 350 nm
• Emission wavelength: 450 nm
 LW 1b: Fluorometric Determination of Quinine in Tonic Water

Reagents and solutions:

• ?? mL, 0.5 M H2SO4 (Couple)
• ?? mL, 100 mg/L in H2SO4 0.5 M, quinine stock solution (Couple)
o Calculate exact concentration to use it as standard solution
 LW 1b: Fluorometric Determination of Quinine in Tonic Water

Tonic water preparation:

• Pipette 0.25 mL of degasified tonic water into 10 mL volumetric flask
• Dilute with H2SO4 0.5 M
• Prepare three samples
 LW 1b: Fluorometric Determination of Quinine in Tonic Water

External calibration:
• Calibration solutions:
o Clean carefully and rinse volumetric flask with small volumes of
H2SO4 0.5 M solution
o Quinine stock solution (0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/L)
o Dilute to 25 mL with 0.5 M H2SO4
 LW 1b: Fluorometric Determination of Quinine in Tonic Water

External calibration:
• Calibration curve construction at λex: 350 nm/ λem: 450 nm:
o Clean and homogenize the sample cell
o Make fluorescence intensitymeasurement (3 replica for
solution)
o Make calibration curve
- Statistical treatment
 LW 1b: Fluorometric Determination of Quinine in Tonic Water

External calibration:
• Measurement of the tonic water sample at λex: 350 nm/ λem: 450 nm:
o Clean and homogenize the sample cell
o Make fluorescence intensity measurement (3 replica for
solution)
• Calculate the amount of quinine
 LW 1b: Fluorometric Determination of Quinine in Tonic Water

Results and reports:

• Calibration:
o External calibration (the most reliable regression line)
o Uncertainty calculation of the concentration obtained by the
regression line
• Sample concentration:
o The analyte’s final concentration at 95% of confidence
o Comparison of results (among couples and/or real value)