66610 - Bioanalytical Chemistry

Learning outcomes

At the end of the course the student will know the basics of conventional spectroscopic and separative analytical techniques, as well as the main bioanalytical methods. He will be able to select the most appropriate analytical method to solve a given analytical question, to apply an analytical protocol and to analyze and interpret analytical results using statistical methods.

Course contents

- The analytical process.

- Statistical treatment of analytical data with elements of chemometrics: errors in quantitative analysis, application of statistics to small sets of data, statistical tests of significance (t-Student, F, Q), propagation of errors, significant digits, statistics of linear relations.

- Parameters of validation of an analytical method: precision, accuracy, sensitivity, limit of detection, robustness.

- The analytical signal and introduction to instrumental methods of analysis.

- UV-Vis spectrophotometry: the electromagnetic spectrum and radiation/matter interaction, UV-Vis absorption (organic and inorganic molecules, transition metal complexes), Beer-Lambert law and limits of validity, spectrophotometric error, quantitative analysis by calibration curves and the standard additions method, analysis of mixtures, components of UV-Vis spectrophotometers, instrumental configurations.

- Fluorimetry: basics on fluorescence excitation and emission spectra, fluorescence-based quantitative analysis, instrumentation, analytical methods based on fluorescence polarization and time resolved fluorescence.

- Principles and application of separative methods: classification of methods, theoretical aspects of chromatography, liquid chromatography (HPLC), main detectors (light absorption, fluorescence, refractive index, light scattering, electrochemical), gas-chromatography (GC), development and optimization of a chromatographic method, coupling of HPLC and GC to mass spectrometric techniques.

- Electrochemical methods: basics on electrochemical processes, Nernst equation, potentiometric measurements, classical and ion-selective electrodes, glass electrode for pH measurement, potentiometric biosensors based on enzymatic reactions.

- Theoretical principles and applications of biospecific methods of analysis.

- Enzymatic methods: principles of enzyme kinetics, Michaelis-Menten equation, determination of catalytic activity of enzymes by simple and coupled enzymatic reactions, one-point and two-point methods, determination of metabolites via end-point methods based on simple and coupled enzyme reactions, spectrophotometric, fluorimetric and non-spectroscopic methods.

- Immunological methods: kinetic and thermodynamic aspects of immunological methods, quantitative competitive and non-competitive immunological methods, homogeneous and heterogeneous methods, synthesis of hapten-protein derivatives for the production of antibodies, production and characterization of mono- and polyclonal antibodies, immobilization of antibodies, synthesis of hapten-enzyme derivatives and of fluorescent and chemiluminescent tracers, different detection techniques (colorimetry, fluorescence and chemiluminescence), development and optimization of an immunological method.

- Laboratory experiments with analysis of unknown samples: quantitative spectrophotometric determination of iron after sample purification by SPE extraction, quantitative spectrophotometric determination of phospholipids by enzymatic method, determination of progesterone by competitive solid-phase enzyme immunoassay, construction and evaluation of a ion-selective electrode for ammonium ion and of an electrochemical biosensor for urea, quali-quantitative analysis of  pharmaceuticals by HPLC.

Readings/Bibliography

The slides used for lectures, detailed protocols for practical laboratory exercises and tracks for laboratory reports will be provided.

Books:

Skoog, West, Holler, Crouch – Fondamenti di chimica analitica - 2° Edizione – EdiSES (2004)

Harris - Chimica analitica quantitativa - 2° Edizione – Zanichelli (2005)

Spandrio - Principi e Tecniche di Chimica Clinica - Piccin Editore (2000)

Teaching methods

Lectures and individual practical laboratory exercises.

Assessment methods

The learning assessment takes place through final oral examination, which ensures the achievement of the following learning objectives:

 - Knowledge of procedures for processing and evaluation of experimental data;

 - Knowledge of the basic principles and procedures of the most important instrumental analytical and bioanalytical techniques;

 - Ability to establish the most suitable bioanalytical technique for solving a particular analytical problem.

An integral part of the exam is constituted by the attendance to individual practical laboratory exercises and the preparation of the laboratory reports. Laboratory tests include the analysis of unknown samples.

The final grade for the course of Bioanalytical Chemistry is defined according to the discussion of laboratory reports, the evaluation of quantitative results obtained for unknown samples and the results of the oral examination.

Teaching tools

The lectures will be conducted by video projector. The practical laboratory exercises will be carried out in university laboratories equipped with the all the necessary instrumentation.

Office hours

See the website of Aldo Roda