Pharmaceutical Biotechnology

Activity type

Discipline fondamentali applicate alle biotecnologie

SSD

CHEM-03/A

Year

1st year

Semester

1st semester

CFU

3

Hours distribution

24 classroom hours

Lecturers

ANNA TROIANI

Activity type

Discipline fondamentali applicate alle biotecnologie

SSD

CHEM-05/A

Year

1st year

Semester

1st semester

CFU

6

Hours distribution

48 classroom hours

Lecturers

ALESSIA CIOGLI

General objectives
The Physical Methods in Organic Chemistry and Radiochemistry course aims to provide students with fundamental knowledge of modern chromatographic, spectroscopic and mass spectrometric techniques, commonly used in the study of organic molecules both in the research field and in control laboratories. The course also aims to provide the ability to identify the most suitable chromatographic techniques for solving real problems, and to understand UV, IR, MS and NMR spectra of organic molecules of biotechnological interest. Basic knowledge of nuclear chemistry, preparation and use of radiopharmaceuticals for diagnostic and therapeutic purposes are also provided. At the end of the course, students will acquire the skills to analyse NMR, IR and MS spectra, to derive the structure of unknown compounds from their combined analysis, and to predict the spectroscopic properties of new compounds.

Specific objectives
1. Knowledge and understanding
The student knows and understands the fundamentals of modern chromatographic techniques: adsorption, partition, thermodynamic aspects, van Deemter equation, composition and morphology of stationary phases, simple structure-retention relationships, solute-stationary phase-mobile phase interaction. The student knows the different elution modes in liquid chromatography (NP, RP, HILIC, PIC-LC, HIC). The student also knows and understands the fundamentals of spectroscopic techniques: matter-radiation interaction, the electromagnetic spectrum, wavelength, frequency, energy content, radiation intensity, absorption, emission, scattering, excited states, quantization. The student knows and understands the theoretical principles and practical applications of IR spectroscopies (harmonic oscillator, anharmonic oscillator, fundamental vibrations, overtone, combination bands, characteristic absorptions of the main functional groups), 1H-NMR and 13C-NMR (nuclei in a magnetic field, resonance, relaxation processes, screen and screen constants, homo- and hetero-nuclear spin-spin coupling, spin systems and Pople notation, Karplus relation). The student knows and understands the main ionization and fragmentation processes underlying the different mass spectrometry techniques; he/she knows the main ion sources and mass analysers suitable for the study of organic molecules and biological macromolecules. He/she is also able to interpret the mass spectrum of model compounds of pharmaceutical and biotechnological interest. The student knows and understands the theoretical principles and practical applications of coupled instrumental techniques (LC-MS), and can understand how the spectral parameters can be influenced by the experimental conditions (physical state of the sample, concentration, solvent, temperature). The student will also be able to determine the decays of unstable isotopes, and the type of radiation emitted, the main radiopharmaceuticals used in diagnostics and therapeutics in relation to the different districts of the organism.
2. Applying knowledge and understanding
The student is able to select the most suitable chromatographic technique based on the structure of the compounds to be analysed and is able to describe the process for the choice of stationary phases, mobile phases and detectors. He/she is able to control and optimize the kinetic and thermodynamic parameters of the chromatographic process and is able to apply the acquired knowledge to new problems typical of research contexts and in the workplace. The student is able to interpret IR, NMR, MS spectra of simple pure organic compounds, and is able to choose the spectroscopic technique or the combination of more techniques suitable for the different structural investigations (control of the conversion of functional groups, identification of impurities). The student is able to apply the known instrumental techniques to new problems that may arise in research or work contexts. At the end of the course the student will know the decay mechanisms and the radiation emitted by a radioelement and the main radiopharmaceuticals used in nuclear medicine, both in diagnostics and therapy.
3. Making judgement
The student is able to broaden and to extend the knowledge acquired during the Master's Degree course with skills relevant to the pharmaceutical aspects that characterize the Specialist Degree Course. The student will be able to independently select the proper analytical method for a specific analytical problem. He/she will also be able to acquire from databases and understand multispectral data useful for solving typical problems in research and production areas such as synthesis laboratories, quality control of active ingredients labs, laboratories of natural products analysis, as well as analysis of complex mixtures and metabolites. These skills are particularly stimulated and developed by carrying out spectral interpretation exercises during lectures and tutorials..
4. Communication
The student will be able to communicate what he/she has learned in a clear and rigorous manner, both to non-expert talkers and to experts in the field. The student is stimulated to interpersonal communication during lectures and classroom exercises.
5. Learning skills
The student will have developed autonomous learning skills related to chromatographic, spectroscopic and spectrometric techniques through the consultation of databases, bibliographic material and scientific literature available online.

Educational objectives: to provide the theoretical basis of chromatographic techniques and of the main spectroscopies. To introduce the student to the use of these methods in the analysis of mixtures and in the determination of the structure of simple organic compounds.

For an easy understanding of the topics covered in the course, it is recommended to review your knowledge of organic chemistry.

PART 1 (6CFU)
High-performance chromatographic techniques. Theory and principles. Main application fields of chromatography. Chromatographic parameters (capacity factors, selectivity, efficiency, resolution). Van Deemter equation. Chromatographic supports and their physico-chemical properties. Bonded-phases: chemistry and stability. Separation mechanisms. UHPLC: General aspects. UHPLC stationary phases. Stereoselective and enantioselective separations. Direct and indirect approach. Chiral stationary phases (CSPs) for HPLC.

Nuclear MAgnetic Resonance (NMR). General aspects. Theory of Nuclear Magnetic Resonance (NMR). Nuclear magnetic moment, spin number, angular momentum, magnetogyric ratio. Larmor precession. Relaxation processes: spin-lattice and spin-spin. Proton Magnetic Resonance Spectrometry (1H-NMR). Important concepts and parameters in NMR. Instrumentation and sample handling. Chemical shift (diamagnetic anisotropy, bond anisotropic effect, ring-current effect, hydrogen bond, solvent polarity and magnetic anisotropy). Spin-spin coupling, multiplets, spin systems. Protons on heteroatoms. Exchangeable protons. Coupling of protons to other important nuclei. Chemical shift equivalence. Magnetic equivalence (spin-coupling equivalence). AMX, ABX, and ABC rigid systems with three coupling constants. Chirality. Vicinal and geminal coupling in rigid systems: Karplus correlations. Long-range coupling. Selective spin decoupling. Double resonance. Nuclear Overhauser Effect (NOE). Shift reagents. Carbon Magnetic Resonance Spectrometry (13C-NMR). Total spin decoupling spectra. Off-resonance spectra. Chemical shift equivalence. Correlation NMR spectrometry. DEPT and APT experiments. Variable temperature NMR. Basics of 1H-NMR and 13C-NMR spectrum interpretation.

Infrared Spectroscopy (IR)
General aspects. Theory of Infrared Spectroscopy (IR). Main functional groups and typical IR absorptions of organic molecules. Basics of Fourier Transform IR (FT-IR) spectrum interpretation. Identification and structure elucidation of organic molecules by interpretation of FT-IR spectra.

Ultraviolet Spectroscopy (UV)
General aspects. Theory of Ultraviolet Spectroscopy (UV). Lambert-Beer law. Electronic transition σ > σ*, n > π*, π > π*. UV instrumentation and solvents. Typical UV absorbing of compounds featuring only bonds, of aliphatic compounds with n electrons and of compounds with electrons. Aromatic systems.

Suggested textbook for spectroscopic techniques in organic chemistry:

1) Silverstein, Webster, Kiemle. Spectrometric Identification of Organic Compounds, 8th Edition.
2) Additional material provided by the teacher

Suggested textbook for mass spectrometry lessons:

1) Textbook:“Identificazione spettroscopica di composti organici” R.M. Silverstein, F.X. Webster Casa Editrice Ambrosiana
2) E. De Hoffmann, J. Charette, V. Stroobant "Mass Spectrometry Principles and Applications" John Wiley & Sons
3) Alison E. Ashcroft "Ionization Methods in Organic Mass Spectrometry" RSC

Suggested textbook for radiochemistry lessons:
F. Cacace “Principi di Chimica Nucleare e Radiochimica”.

Module:
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Module:
N/D

Lessons will be held in person or in mixed mode according to the anti-covid provisions in force.

Attendance at the courses is compulsory.
For an easy understanding of the topics covered in the course, it is recommended that you have passed the Organic Chemistry I.

The written test focuses on the structural elucidation of an organic molecule starting from 1H-NMR, 13C-NMR, FT-IR spectra.
The oral exam will give more attention to the chromatographic part and to the theory.

Module:
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Module:
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• High Performance Liquid Chromatography (HPLC). General aspects.
  Chromatographic parameters (capacity factors, selectivity, efficiency,
  resolution). Van Deemter equation. 


• Bonded-phases: chemistry and stability.Separation mechanisms(NP, RP, SEC, IP-RP, HILIC, HIC)


• Deterctor for HPLC (2h)


• Chiral stationary phases (2h)


• Theory of Nuclear Magnetic Resonance (NMR). Nuclear magnetic moment,
  spin number, angular momentum, magnetogyric ratio. Larmor precession.Proton Magnetic Resonance Spectrometry (1H-NMR). Important
  concepts and parameters in NMR. Instrumentation and sample handling.


• Chemical shift (diamagnetic anisotropy, bond anisotropic effect,
  ring-current effect, hydrogen bond, solvent polarity and magnetic anisotropy). 


• Spin-spin coupling, multiplets, spin systems.AMX, ABX, and ABC rigid systems with three coupling constants. 


• Analysis of spectra


• Vicinal and geminal coupling in rigid systems: Karplus correlations.
  Long-range coupling. Protons on heteroatoms. Exchangeable protons. 


• Coupling of protons to other important nuclei. Chemical shift
  equivalence. Chirality.


• Carbon Magnetic Resonance Spectrometry (13C-NMR). Total spin
  decoupling spectra. Off-resonance spectra.


• Theory of Infrared Spectroscopy (IR). Main
  functional groups and typical IR absorptions of organic molecules. Basics of
  Fourier Transform IR (FT-IR) spectrum interpretation. Identification and
  structure elucidation of organic molecules by interpretation of FT-IR spectra. 


• Theory of Ultraviolet Spectroscopy (UV).
  Lambert-Beer law. Electronic transitions s→s*, n→s*, n→p*, p→p*. UV
  instrumentation and solvents. Typical UV absorbing of compounds featuring only s bonds, of aliphatic compounds with n electrons and of compounds with p electrons. Aromatic systems. 


• Interpretation of all spectral data


• Week 1
  Introduction to the course: review of atomic structure; chemical mass unit;
  mass defect; nuclear stability.


• Week 2–3Nuclear
  stability and instability: stability map and magic numbers; proton-rich
  and neutron-rich nuclei; nuclear decay processes: alpha decay, beta⁻ decay, beta⁺ decay, and electron capture;
  stability criteria; interaction of radiation with matter; kinetics of
  radioactive decay; nuclear reactions and the compound nucleus; classification
  and energetics of nuclear reactions; introduction to radiopharmaceuticals and nuclear
  syntheses.


• Week 4 Radioisotopes in diagnostics


• Week 5 Radiopharmaceuticals


• Week 6–8 Introduction
  to mass spectrometry and analytical chemistry: The mass spectrometer and the
  mass spectrum: base peak, molecular and pseudomolecular ions, fragmentation;
  ionization sources (EI, CI); mass analyzers and resolution; the nitrogen rule;
  isotopic multiplets and isotopic distribution; exercises on isotopic peak
  deconvolution; main fragmentation patterns and characteristic features of
  functional groups; exact mass and accurate mass; resolution.


• Week 9–12 Mass
  spectrometry applied to macromolecules:Atmospheric pressure ionization (ESI and
  APCI); formation of multiply charged ions; determination of molecular mass from
  multiply charged ions; determination of charge state from the distance between
  ¹²C and ¹³C peaks; solid-state ion sources: MALDI ionization; tandem mass
  spectrometry and collision-induced dissociation; overview of amino acid
  fragmentations; applications of mass spectrometry to tissue imaging techniques;
  applications of mass spectrometry in the study of peptides and
  proteins.Laboratory exercise:Numerical analysis of mass spectra; general
  review.