Jan 23, 2023
Solid fungal extraction and C8 reversed phase chromatography
- 1University of Auckland
Protocol Citation: Shara Van De Pas, Siouxsie Wiles 2023. Solid fungal extraction and C8 reversed phase chromatography. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1onpklr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 27, 2022
Last Modified: January 23, 2023
Protocol Integer ID: 71945
Keywords: C8 silica, chromatography
Abstract
Protocol for freeze-drying fungal cultures and preparing extracts using C8 reversed-phase chromatography.
Materials
Plasticware
| Description | Catalogue number | Supplier | |
| 90mm Petri Dishes | LAB-021MR | Medi'Ray |
Solvents, etc
| Description | Catalogue number | Supplier | |
| Methanol LR | 32108-5L | Sigma Aldrich | |
| Dichloromethanol (DCM) | 21409-4L | Sigma Aldrich | |
| D4 Methanol | DLM-24-10 | Sigma Aldrich | |
| C8 silica gel | LU013BM | Luknova |
Equipment
- Sterile scalpel handle and blade
- Freeze dryer (Labogene)
- Rotary evaporator (Buchi)
Safety warnings
Solvent hazard classes:
Dichloromethane (DCM): 6.1(3)
Hexane: 3(2)
Ethyl acetate: 3(2)
Methanol: 6(6.1)
Crude extraction of fungal compounds
Crude extraction of fungal compounds
Subculture fungus onto ~40 Petri dishes of media of choice and seal the plates with parafilm. Grow at the appropriate growth temperature until the fungus reaches the required age or coverage.
Once grown, freeze plates overnight (-20 °C )
Freeze dry over two days (-80 °C ) until plates are dry and crisp.
Weigh a clean 500ml beaker and break up dry fungal tissue into small pieces. Reweigh the beaker to obtain the dry weight of the fungus.
Submerge dry fungal tissue in methanol (MeOH) for 4 hours.
Filter into a 500ml round bottom flask.
Dry at reduced pressure on a rotary evaporator at 130 mbar.
Re-submerge fungal tissue in dichloromethane (CH2Cl2), cover with tinfoil and leave to soak overnight.
The following day filter into the same round bottom flask and dry at 500 mbar.
C8 reversed phase chromatograpahy
C8 reversed phase chromatograpahy
Remove some crude product from the flask for downstream biological testing.
Dissolve the remaining crude product in 10%M MeOH before loading onto C8 reversed-phase silica gel. Elute with a gradient of H20:MeOH (25% increments of 100ml volume) to afford five fractions (F1-F5). Apply pump pressure and collect the fractions in 250ml round bottom flasks.
Schematic of C8 reversed-phase chromatography
Dry the 5 fractions at reduced pressure on the rotary evaporator at 130mbar (MeOH) and 40mbar (Water).
NMR and Samples for Biotesting
NMR and Samples for Biotesting
When flasks are dry add 0.35-0.4ml of D4 MeOH to each flask (Fraction 1 isn't necessary as it will contain sugars and salts) and mix. Using a 1ml syringe and needle draw up the methanol-fungi mixture into the syringe and add it to a clean, labelled NMR tube.
Cap samples gently and load them on to the AvanceIII-400mhz instrument.
Name each of the samples and use these settings:
- Proton H+
- 64 scans
- Solvent: d4 Methanol
Label and weigh 5 empty biotesting vials for further testing. Add the contents of the NMR tubes to each of the tubes and allow to dry. Reweigh the dry tubes and label them with the mass (in milligrams). Parafilm the lids and send back for extract testing.