Industrial pharmacy

ALESSIA CIOGLI

General expected learning outcomes
This course is designed to provide qualitative identification of organic compounds and mixtures, with emphasis on advanced techniques of separation, purification and spectral identification.
The student will experience the most important spectroscopic and analytical techniques used for structure elucidation, which will be useful for his employment in regulatory affairs and healthcare industries.

Specific expected learning outcomes
Understanding and interpretation of UV, FT-IR, proton and carbon-13 NMR spectra.
Structural elucidations of simple organic compounds starting from their raw formula and spectroscopic data. Starting from a mixture of a few known compounds, propose the most suitable chromatographic method based on the acquired knowledge.

Aims: understanding and interpretation of UV, FT-IR, proton and carbon-13 NMR spectra. Structural elucidations of simple organic compounds starting from their raw formula and spectroscopic data. Starting from a mixture of a few known compounds, propose the most suitable chromatographic method based on the acquired knowledge.

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

High-performance chromatographic techniques.
Theory and principles. Main application fields of chromatography. High Performance Liquid Chromatography (HPLC). General aspects. Chromatographic parameters (capacity factors, selectivity, efficiency, resolution). Van Deemter equation. Chromatographic supports and their physico-chemical properties: surface chemistry, specific surface area, pore size and volume. Bonded-phases: chemistry and stability. Separation mechanisms: liquid-liquid, liquid-solid, normal-phase (NP), reversed-phase (RP). Ion-Exchange Chromatography (IEC). Paired-Ion Chromatography (PIC). Size-Exclusion Chromatography (SEC). Hydrophobic-Interaction Chromatography (HIC), Hydrophilic interaction Chromatography (HILIC). Elution mode: isocratic and gradient. Detectors.
UHPLC: General aspects. UHPLC stationary phases.
High-Resolution Gas Chromatography (HRGC). General aspects. Microbore and packed microcapillary columns.
Stereoselective and enantioselective separations. Direct and indirect approach. Main interactions between selector and selectands. Thermodynamics of enantiomers separation. Chiral stationary phases (CSPs) for HPLC and HRGC.

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 transitions →*, n→*, n→*, →*. 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.
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.
Mass Spectrometry (MS)
Ionization systems: electron impact, chemical ionization, fast atom bombardment (FAB) – ESI – APCi – MALDI.
Analyzers: magnetic and electrostatic sectors, quadrupolar systems, ion trap, time of flight systems, orbitrap, tandem mass spectrometry.
Molecular ion, exact masses, isotopic abundance, isotopic clusters, elemental composition.
Typical fragmentation patterns in mass spectra, McLafferty rearrangement.

Silverstein, Webster, Kiemle.Spectrometric Identification of Organic Compounds, 8th Edition.

Lessons will be held in person.

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.

Questions:
- Van Deemter equation
- PIC-RP-HPLC.
- UV spectra of alfa-beta unsaturated ketones
- NOE effect and 13C-NMR

• High-performance
  chromatographic techniques.
  
  Theory and
  principles. Main application fields of chromatography. 


• High Performance Liquid Chromatography (HPLC). General aspects.
  Chromatographic parameters (capacity factors, selectivity, efficiency,
  resolution).


• Equation di Van Deemter. (2h) 


• Chromatographic supports and their physico-chemical properties: surface
  chemistry, specific surface area, pore size and volume. 


• Bonded-phases: chemistry and stability, synthesis of C18 stationary
  phases. Separation mechanisms: normal-phase (NP), reversed-phase (RP). 


• Ion-Exchange Chromatography (IEC). Paired-Ion Chromatography (PIC).
  Size-Exclusion Chromatography (SEC). Hydrophobic-Interaction Chromatography
  (HIC), Hydrophilic interaction Chromatography (HILIC). 


• Elution
  mode: isocratic and gradient. Detectors (UV, PDA, RI, ELSD, MS).


• UHPLC:
  General aspects. UHPLC stationary phases. 


• High-Resolution
  Gas Chromatography (HRGC). General aspects. Microbore and packed microcapillary
  columns. 


• Stereoselective
  and enantioselective separations. Direct and indirect approach. Main
  interactions between selector and selectands. Thermodynamics of enantiomers
  separation. Chiral stationary phases (CSPs) for HPLC and HRGC.


• 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


• Instrument and sample preparation


• Ultraviolet Spectroscopy (UV)
  
  General
  aspects. 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. 


• 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. AMX, ABX, and ABC rigid systems with three coupling constants.
  Chirality. 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. Magnetic equivalence (spin-coupling
  equivalence). Chirality. 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.


• 2D-NMR: basic experiments


• Variable temperature NMR. D-NMR vs D-HPLC


• Basics of 1H-NMR and 13C-NMR spectrum
  interpretation.


• Mass Spectrometry (MS)
  
  Ionization
  systems: electron impact, chemical ionization, fast atom bombardment (FAB) –
  ESI – APCi – MALDI.
  
  Analyzers:
  quadrupolar systems, ion trap, orbitrap. HPLC-MS: ESI and APCI sources coupled
  with HPLC. Molecular ion, exact masses, isotopic abundance, isotopic clusters.
  
  Typical
  fragmentation patterns in mass spectra, McLafferty rearrangement.