Course code:
257B2
Course name:
Advanced Structural Instrumental Methods

Academic year:

2024/2025.

Attendance requirements:

There are no requirements.

ECTS:

6

Study level:

graduate academic studies

Study program:

Biochemistry: 1. year, winter semester, elective (E51B2) course

Teacher:

Ljubodrag V. Vujisiæ, Ph.D.
associate professor, Faculty of Chemistry, Studentski trg 12-16, Beograd

Assistants:

Gordana B. Krstiæ, Ph.D.
assistant professor, Faculty of Chemistry, Studentski trg 12-16, Beograd

Ivana V. Sofreniæ, Ph.D.
assistant professor, Faculty of Chemistry, Studentski trg 12-16, Beograd

Hours of instruction:

Weekly: two hours of lectures + two hours of labwork (2+0+2)

Goals:

Students should get acquainted with the following areas of instrumental methods relevant to biochemistry:

IR spectroscopy, ATR and IR Imagining of biological samples (Determination of protein secondary structure, protein-protein and protein-ligand interactions. Analytical profiling of complex mixtures, e.g. propolis).

Multipulse 1D (1H, 13C, 15N and 31P), 2D, 3D and 4D NMR methods (Determination of primary, secondary and tertiary structure of proteins and protein – ligand interactions. Analytical profiling of complex mixtures, e.g. blood and urine samples. Medicinal diagnostics).

Mass spectrometry (MS), Tandem mass spectrometry (MS/MS) and multistage mass spectrometry (MSn) and MALDI imaging in biomedical sample analysis (Determination of protein structure. Analytical profiling of complex mixtures e.g. tissue).

Liquid chromatography combined with mass spectrometry and NMR spectroscopy (LC/MS, LC/MS/MS, LC/MSn, LC/NMR. Targeted and non-targeted analysis).

Outcome:

Students will be trained for processing the recorded spectra and solving the structures of organic compounds using multipulsed NMR experiments and modern mass spectrometry methods. Moreover, students will be able to prepare and profiling samples of complex mixtures in various matrixes by advanced instrumental methods.

Teaching methods:

Lectures, labwork, homework, colloquia, and semester papers.

Extracurricular activities:

Guest lectures.

Coursebooks:

Main coursebooks:

  1. Slobodan Milosavljević: Strukturne instrumentalne metode
  2. Vele Tešević: Osnove masene spektrometrije organskih jedinjenja
  3. Ernö Pretsch, Philippe Bühlmann and Martin Badertscher: Structure determination of organic compounds, Tables of Spectral Data
  4. L. D. Field, S. Sternhell, J. R. Kalman: Organic structures from spectra, fourth edition

Supplementary coursebooks:

  • Recent scientific papers.

Additional material:

  Course activities and grading method

Lectures:

0 points (2 hours a week)

Syllabus:

1. NMR spectroscopy

  • Multipulse 1D NMR methods (INEPT, APT, DEPT, NOE, STD - saturation transfer difference, T1 and T2 measurement).
  • Two-dimensional (2D) NMR methods (2DJ resolved NMR, COSY, TOCSY, HSQC, HMBC, NOESY, ROESY, DOSY) and their application for biomolecule analysis.
  • Three-dimensional (3D) and four-dimensional (4D) NMR methods and their application for determining the structure of biopolymers - proteins.
  • Detection of 15N and 31P NMR spectra and their application in biochemistry.
  • Protein-ligand screening by NMR.
  • Analytical profiling of samples in complex matrixes (e.g. blood and urine by NMR).
  • Liquid chromatography combined with NMR spectroscopy (LC/NMR).

2. IR spectroscopy

  • Determination of protein secondary structure and spectra deconvolution.
  • Determination of protein-protein and protein-ligand interactions using IR spectroscopy.
  • IR imaging of biomedical samples.

3. High-performance thin-layer chromatography (HPTLC). Bioautography assay.

4. Mass spectrometry

  • Ionic sources (EI, CI, FAB, SIMS, DART, MALDI, SELDI, ESI, APCI, APPI, CID/CAD).
  • Ion Separation - Analyzers (Ion mobility, B, E, Q, Ion Trap (IT), TOF, ICR (MR MS), Orbitrap).
  • Tandem mass spectrometry (MS/MS or MS2) and multistage mass spectrometry (MS/MS/MS... MSn).
  • MS/MS experiments of qualitative and quantitative targeted analysis: Product Ion Scan, Daughter Ion Scan, Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), and Neutral loss.
  • Determination of protein structure by MS/MS methods.
  • MALDI imaging in biomedical sample analysis.
  • Liquid chromatography coupled with mass spectrometry (LC/MS, LC/MS/MS, LC/MSn).
  • Instrumental methods in chemical weapons analysis.

Labwork:

10 points (2 hours a week)

Syllabus:

  1. Structure elucidation of organic compounds based on H,H-COSY, HETCOR, DEPT, 2DJ, H,H-NOESY NMR spectra.
  2. Structure determination of organic compounds and proteins using MS, MS/MS and MSn methods.
  3. Introducing existing equipment of the Faculty of Chemistry to the students (IR, GC, LC, HPTLC, MS, GC/MS, LC/MS, NMR); sample preparation, spectra capture and interpretation.
  4. Software training in SPECTRAL, OMNIC, CHEMSTATION, COMPAS, TOPSPIN, MESTRE NOVA, and SIMCA.
  5. Antioxidative activity (DPPH radical assay) of various sample types.
  6. HPTLC - Bioautography assay.
  7. Methods for fraud detection in food supplements.
  8. Secondary structure determination of proteins using FTIR spectroscopy.
  9. STD NMR spectroscopy. Protein-ligands screening.

Semester papers:

20 points

Colloquia:

10 points

Written exam:

20 points

Oral exam:

40 points