Principle of spectroscopy and applications to quantitative biology

A.Y. 2021/2022
Overall hours
Learning objectives
The primary objective of this course is to introduce the basic concepts, principles, and techniques of modern analytical chemistry that would provide students with an analytical mind shape and abilities to solve diverse analytical problems, involved in the biotechnologies, in an efficient, quantitative and qualitative way. One of the aim is to provide to the students adequate preparation in order to identify the different components of the biological system and to convey the importance of accuracy and precision of the analytical results.
Expected learning outcomes
On successful completion of this course, students will be able:
1) to understand the different analytical methods for molecular and supramolecular analysis.
2) to establish an appreciation of the role of chemistry and molecular spectroscopy in quantitative analysis, in measurement and problem solving for analytical tasks.
Moreover students will be able to select molecular probes and to design experiments in order to understand the mechanism of cellular uptake, or to better visualize the different components of biological system.
Through the experimental part of the course, the students will be trained to correctly interpret and to communicate the experimental results.
Course syllabus and organization

Single session

Lesson period
First semester
More specific information on the delivery modes of training activities for academic year 2021/22 will be provided over the coming months, based on the evolution of the public health situation.
Course syllabus
Principles of spectroscopy and applications to quantitative biology
The topics are:
Classification of analytical methods
Types of instrumental methods
Selecting an Analytical Methods
Evaluation of analytical data: precision and accuracy, method of least squares

An introduction to electroanalytical chemistry
Electrochemical cells
Cell potentials
Electrode potentials
Calculation of cell potentials from electrode potentials
Types of electroanalytical methods

Potentiometric methods
Reference electrodes
Metallic indicator electrodes
Membrane ion-selective indicator electrodes
Junction potentials
Instrument for measuring cell potentials and calibration methods,
Direct potentiometric measurements
Potentiometric titrations

Spectrophotometric methods
Properties of electromagnetic radiation: an overview
Quantum mechanical properties of radiation and light/matter interaction
Molecular ultraviolet/visible absorption spectroscopy: terms employed in absorption spectroscopy
Quantitative aspects of absorption measurements: the Lambert-Beer equation
The absorbing species
Application of absorption measurements to qualitative analysis
Spettrophotometric titrations
Instrument components for absorption measurement in the UV-Vis region.

Molecular Emission spectroscopy
Theory of fluorescence and phosphorescence: photophysical deactivation paths of the excited-state
Kinetics of excited states quenching
Steady state and time-resolved emission spectroscopy: quantum yield and lifetime measurement.
Instruments for measuring fluorescence and phosphorescence
Applications and photoluminescence methods in quantitative analysis.
Luminescent probes for cell imaging.

Infrared absorption spectroscopy
Theory of infrared absorption: fundamental principles.
Frequency of infrared absorption and chemical structure.
Infra-red Spectrophotometer and Fourier Transform IR (FT-IR).
Qualitative applications of infra-red absorption.
Quantitative applications of IR spectroscopy.
Nuclear magnetic resonance (NMR)
General principles, nuclear properties, magnetic field and electromagnetic radiation (radiofrequency).
Proton nuclear resonance: 1H NMR chemical shift and structure, chemical shift equivalence: random, isochronal protons, homotopic protons, enantiotopic, diasterotopic.
Magnetic Equivalence. Practice on a collection of proton spectra of simple aromatic compounds, spin system simulation (cascade diagrams), reading of spin systems (determination of coupling constants and chemical shift).
NOe differential experiments with examples.
Nuclear magnetic resonance of carbon-13: general principles, relative and absolute sensitivity of an NMR experiment, chemical shifts, proton-proton and proton-carbon coupling, examples of simple aromatic and non-aromatic compounds, 13C-NMR spectra recorded with the APT and DEPT techniques.
Two-dimensional magnetic resonance spectroscopy (2D). Correlation Spectroscopy COSY.

Chromatographic separation methods
A general description of chromatography
Migration rates of species
Band broadening and column efficiency
Optimization of column performances
Applications of chromatography
Gas-chromatography: principles of gas-liquid chromatography
Instruments for gas-chromatography
Gas-chromatographic columns and stationary phases
High-performance liquid chromatography: scope of HPLC
Column efficiency in liquid chromatography
Instruments for liquid chromatography
Thin-layer chromatography.

Mass Spectrometry
General principles and instrumentation.
Sources (EI, CI, FAB, MALDI, ESI APCI), analyzers (magnetic, quadrupole, ion trap), detectors.
Identification of pure compounds by mass spectrometry
Prerequisites for admission
Knowledge of the fundamentals of the general and inorganic chemistry, organic chemistry, physics and mathematics.
Teaching methods
The course is divided into classroom lectures, in which the topics are illustrated both with slides and by carrying out exercises on the blackboard. At the end of the course, an educational experimental laboratory is organized in which the students, organized in small groups, carry out four different experiences concerning both traditional and instrumental quantitative analysis.
Teaching Resources
Skoog, West, Holler, Crouch, Fondamenti di Chimica Analitica. EdiSES. (3° ed. italiana 2015)
Slides of the lessons available on Ariel website of the course
Assessment methods and Criteria
The knowledge of the topics reported in the course's program will be evaluated by means of a three hours-written test, which is aimed at verifying the knowledge of the topics. In this test the student is asked to solve four exercises concerning the evaluation and the interpretation of data collected from instrumental analysis of different molecules. Two or three theory questions concerning analytical approaches for the characterization of analytes should be present in the written test.
Examples of problems and questions and their evaluation will be provided during the course.
CHIM/01 - ANALYTICAL CHEMISTRY - University credits: 0
CHIM/02 - PHYSICAL CHEMISTRY - University credits: 0
CHIM/06 - ORGANIC CHEMISTRY - University credits: 0
Practicals: 48 hours
Lessons: 56 hours
Professor: Panigati Monica