Methods In Natural Product Chemistry Flashcards
Define bioassay-guided isolation
Physical process used to isolate biologically active chemicals from a natural source ie plants , microbes
Discuss preparation and extraction
Identification and classification by field botanist, into species, families and collect from aerial parts ie leaves
Dry : air dried / drying cabinets
Grind to small particles using blender or mill ( do that 2 times ) to get fine powered
Extraction - cold extraction , soxhlet extraction
For extraction describe 3 types of exctraction
- Cold extraction- biomass extracted using solvent of increasing polarity ke hexane, chloroform, ethyl acetate
- Soxhlet - biomass placed in soxhlet thimble through which solvent is continuously refluxed , and contents emptied into round bottom flask once the solvent reaches certain level
- Supercritical fluid- some gases behave as liquids when under pressure and solvating properties
Give examples of isolation methods
- Partionining
- Gel chromatography
- Ion-exchange chromatography
- Flash
- Thin layer
- High performance liquid chromatography
Gel chromatography
Used as initial clean up step employs cross linked dextran and the gel is loaded into a column and extract added to top of column . Large molecules are first , followed by smaller ones
Good method for separation of chlorophyll, glycerides that interfere with biological assay
Partitioning
Clean up step , uses 2 immiscible solvents to which extract is added
Steps :
1. Water / light petroleum ether to generate non polar fraction in organic layer
2. Water / chloroform to give medium polar fraction in organic layer
3. Remaining aq layer will contain polar water soluble natural products
Ion -exchange
Limited to products that carry a charge. Stationary phase has charged groups which exchange with ions of functional groups present in natural products as mobile phase moves through sorbent . Ion exchange sorbents divided into 2 groups cationic exchangers ( exhange protons to cations of natural products)
Anionic exchangers (exchange their anions with anions of natural product)
Flash chromatography
Quick
employs prepacked solvent resistant plastic catridges which contain sorbent ie silica.
Extract is dissolved in solvent and loaded into column , solvent is then pumped through column and fractions are collected to result in rapid separation of extract components
Speed minimize contact with reactive sorbents , cartridges may be redused so reduce cost of bioassay guided process
Compounds can be detected by TLC or UV light
Thin layer chromatography
Purify small number of components , typically following flash seperation
this methods uses glass or aluminum plates that are pre-coated with sorbent .
The compound mixture is loaded at 1-2 cm from bottom edge of plate as either a spot or continuous band. Plate is lowered into a tank containing solvent which migrate up the plate and separate compound mixture according to polarity. Detectors employed include :uv light and spray reagents
Advantage and disadvantage of TLC
Adv: a large number of samples may be analyzed and separated simultaneously
Disadvantage: loading and speed are poor compared with flash chromatography
Poor detection and control of elation compared with HPLC
High performance liquid chromatography
Used for analysis and isolation of bio active natural products. Useful for fingerprinting biologically active extracts and comparisons can be drawn with chromatographs and UV spectra stored in electronic library. Uses stationary phase C18 employing water/methanol mixtures as mobile phase
Mobile phase run in gradient elution mode in which concentration of solvent is increased over period of time
Structure elucidation meaning and methods
Determine structures of compounds on micrograms of material
Involve:
1. IR spectroscopy
2. Uv spectroscopy
3. Mass spectrometry
4. Nuclear magnetic resonance
Uv and IR -identify aromatic ring , conjugation
Mass spectrometry
Allows measurement of molecular weight, used to identify molecular ions accurately to ascertain exact number of hydrogens , carbons, oxygens and other atoms present in molecule this gives molecular formula. Electron impact ionization technique give good fragmentation of molecule as fragments can be assigned to functional groups present in compound .
Chemical ionization (CI)
Electron ionization (ESI)
Fast atom bombardment (FAB)
Describe fast atom bombardment
Submit a compound and computer program calculate number of carbon, hydrogen , oxygen atom to give molecular weight. (M-Me corresponds to methyl group , M+H suggest hydrogen ion is picked up , M-H , hydrogen ion is being lost
NMR spectroscopy
Indicate number of hydrogen atoms associated with particular group and how shielded (electron donating)or unshielded (electron withdrawing) that group is
