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 (bio)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

Room lectures

- Statistical treatment of analytical data (6 hours): errors in quantitative analysis, application of statistics to small sets of data, statistical tests of significance, propagation of errors, significant digits.

- Quantitative analysis, calibration and validation (6 hours): calibration curves, construction of a calibration curve, linear and non-linear calibration curves, linearization of calibration curves, principles of regression analysis, linear and non-linear regression, determination coefficient, evaluation of the quality of a calibration curve, calibration methods, validation of an analytical method, characteristics of an analytical method (accuracy, precision, specificity and selectivity, linearity, dynamic interval, LOD, LOQ, robustness).

- Instrumental analytical methods (10 hours). UV-Vis spectrophotometry: the electromagnetic spectrum and radiation/matter interaction, UV-Vis absorption, Beer-Lambert law and limits of validity, spectrophotometric error, quantitative analysis, analysis of mixtures, components of UV-Vis spectrophotometers, instrumental configurations. Fluorimetry: fluorescence and phosphorescence, fluorescence excitation and emission spectra, quantitative analysis, instrumentation. Electrochemical methods: electrochemical processes, Nernst equation, potentiometry, classical and ion-selective electrodes, glass pH electrode, potentiometric biosensors based on enzymatic reactions. Separative methods: classification of methods, theoretical aspects of chromatography, development and optimization of a chromatographic method, liquid chromatography (HPLC) and detectors, gas-chromatography (GC) and detectors, coupling of HPLC and GC to mass spectrometric techniques.

- Bioanalytical methods (10 hours): enzyme kinetics, Michaelis-Menten equation, determination of enzymes by simple and coupled enzymatic reactions, determination of enzyme substratess via end-point methods based on simple and coupled enzyme reactions, detection of products (direct spectrophotometric methods, methods based on chemically- or enzymatically-generated derivatives, fluorimetric methods, non-spectroscopic methods. Immunological methods: kinetic and thermodynamic aspects of antigen-antibody reactions, competitive and noncompetitive immunological methods, homogeneous and heterogeneous immunological methods, production and characterization of mono- and polyclonal antibodies, immobilization techniques, tracers, of antibodies, synthesis of hapten-enzyme derivatives and of fluorescent and chemiluminescent tracers and detection techniques (colorimetry, fluorescence and chemiluminescence), development and optimization of an immunological method.

Laboratory exercises

Laboratory exercises in single place or in pairs with application of analytical (spectrophotometric, potentiometric, and separative) and bioanalytical (enzymatic and immunological methods) techniques treated in the theoretical lessons. The laboratory exercises are quantitative and include the analysis of an unknown sample and the elaboration of the experimental results to obtain its concentration.

Readings/Bibliography

PowerPoint slides of room lectures, supplementary material, instructions for laboratory exercises and for drafting laboratory reports available on “Virtuale” (https://virtuale.unibo.it, access with credentials reserved for students enrolled at the University of Bologna).

- Fondamenti di Chimica Analitica di Skoog e West - J.F. Holler, S.R. Crouch - 3° Ed. (EdiSES, 2015).

- Chimica Analitica Quantitativa - D.C Harris - 3° Ed. (Zanichelli, 2017).

- Principi e Tecniche di Chimica Clinica – L. Spandrio – 1° Ed. (Piccin, 2000).

- Bioanalytical Chemstry – A. Manz, P.S. Dittrich, N. Pamme, D. Iossifidis - 2° Ed. (World Scientific, 2015).

Teaching methods

The course is made up of room lectures and laboratory exercises. During the lectures the topics of the course will be presented and discussed, with theoretical insights and explanatory examples. The laboratory exercises are designed to allow each student to acquire the knowledge of some instrumental (bio)analytical techniques and the necessary skills for working in a laboratory according to quality and safety guidelines. Laboratory exercises include the analysis of unknown samples. The student will process the results obtained in the laboratory using the tools learned in the course and will submit reports with the analysis of the data and the concentration of unknown samples.

Assessment methods

Verification of knowledge on the whole program by means of the following tests.

- Oral exam on the topics covered during the course, aimed at verifying the acquisition of knowledge and expected skills. In particular, the knowledge of procedures for processing and evaluating experimental data and the understanding of the basic principles and procedures of the main analytical and bioanalytical instrumental techniques will be evaluated. Evaluation will be based on the demonstration of the critical understanding of the topics and the ability to discuss them.

- Evaluation of the laboratory exercises conducted based on the reports submitted by the student. For such evaluation, both the correctness of the procedures used for the elaboration of the experimental data and the concordance of the obtained results with the real concentrations of the unknown samples will be considered.

The final grade will consist of the sum of the marks obtained in the oral exam (maximum 20/30) and in the laboratory exercises (maximum 10/30). The submission of the reports according to the modalities that will be agreed during the laboratory is mandatory to be admitted to the final exam.

Teaching tools

Video projector and (if necessary) virtual room for online lessons. Laboratory exercises will take place in an equipped computer laboratory. The teaching material (PowerPoint presentations, text of numerical and laboratory exercises) will be made available to the student in electronic format on “Virtuale” (https://virtuale.unibo.it, access with credentials reserved for students enrolled at the University of Bologna).

Office hours

See the website of Massimo Guardigli