Methods in Natural product chem Flashcards
Explain Bioassay guided isolation
Physical process to isolate biologically active chemicals from their source/biomass
Once identified and classified, the biomass samples are collected, prepared and extracted. Explain the proces.
- Dried- Airdrie, drying cabinets, lyophilizers.
- Grinding- 1st with a course mill then a fine mull to make fine powder
- Extraction- Cold extraction, hot percolating, supercritical fluid extraction, soxhlet extraction (for thermo stable products)
Explain cold extraction
Done using solvents of increasing polarity
e. g hexane, chloroform, acetone, methanol THEN water.
Explain hot Percolation
crushed biomass is stewed using ethanol/it’s mixtures
Explain supercritical fluid extraction
Principle is that some gasses behave as liquids when under pressure and have solvating properties
Soxhlet extraction explain please
biomass n soxhlet thimble
solvent is continuously refluxed through
Soxhlet apparatus empties contents into round-bottomed flask once a certain level is reached
How are solvents removed after extraction?
Extracts are concentrated under vacuum using rotary evaporator
blown down under nitrogen for small volumes, freeze-dried using lypophilizer
After extraction is isolation. What are the 6 isolation methods?
- Partitioning
- Gel chromatography
- Ion-exchange chromatography
- Flash chromatography
- Thin layer chromatography
- High performance chromatography
Gel/size exclusion chromatography, application and use.
This is a non-destructive, soft method with high recovery.
Initial cleanup step
Cross linked dextran
Gel into column, extract added
Large molecules first, then smaller
Used for chlorophylls, FAs, glycerides, large molecules that interfer with biological assay
Partitioning, clean up step too, steps are
Clean up that uses 2 immiscible solvents
-water/hexane to generate non-polar fraction in organic layer
-water/dichloromethane OR water/chloroform OR water/ethyl acetate to give medium-polar fraction in the organic layer
-remaining aq layer will have hydrophylic natural products
Ion-exchange chromatography, uses and how
-Separates small polar compounds in particular ionic natural products
-The sorbet/stationary phase has charged gps and mobile counter ions which may exchange with ions of functional gps present in the natural productas the mobile phase moves through solvent
How are the sorbents in ion exchange divided
°Cation exchangers- have acidic gps, exchange protons with cations of natural products
°Anion exchangers- have basic groups in resins and can exchange their anions with anions from the natural product
Flash chromatography how
Quick and effective
prepacked solvent resistant plastic cartridges contain the sorbent e. g silica
bioactive is dissolved in solvent, then. into column
solvent is pumped through column and fractions are collected resulting in rapid separation of extract components
Speed reduces contact time with reactive sorbent
Cartridges are reusable = cheap
Detect fractions via TLC or UV detector
Thin layer chromatography
how
Popular, easy, cheap, can handle large samples simultaneously
Loading and speed are poor, poor detention and control of election compared with high performance liquid chromatography
for small number of components
done after flash
-glass/aluminum plates pre coated in sorbent, e. g silica gel
Biomass is loaded, 1-2cm from bottom of edge as a spot
lower plate into tank containing predetermined solvent which will migrate up the plate and cause separation v according to polarity
Use UV 254nm spray reagent as detector
HPLC
When used with UV detector e. g photo diode array, you acquire spectrum from 190nm to 800nm
Helps finger print bioactives and comparison can be made between chromatograms and UV spectra stored in electric library
