This method describes how to separate kerogen from siliciclastic rocks and was originally written by Eva Stuëken. The silicate fraction is dissolved and removed, such that the kerogen remains as a solid for further analysis.
Hydrofluoric acid (HF) is extremely corrosive. You must take the EH&S HF course before conducting this procedure. Review the HF Spill Procedure before continuing.
- Protective clothing (apron, gloves, face shield, closed shoes)
- 250ml Teflon bottles
- Teflon tape
- Portable balance
- Concentrated hydrofluoric acid (HF), reagent grade (can be purchased from Chemstore)
- 18 MΩ DI-H2O (referred to as water from hereon)
- Powdered boric acid H3BO3, reagent grade (from Chemstore)
- Weigh boats
- Aluminum foil
- Teflon spatula
- 100ml graduate cylinder, made of plastic (glass reacts with HF)
- Shaking water bath
- Large centrifuge
- Two acid waste containers (HCl, HF + H3BO3). HF waste must be in plastic, not glass!
- Optional: concentrated hydrochloric acid (HCl), reagent grade
Weighing out the powder
- Wrap Al foil around the upper part of an empty, uncapped Teflon bottle. The foil will reduce electrostatic jumping of rock powder.
- Place the small portable balance into the fume hood
- Tare with empty Teflon bottle (empty weight of bottle not needed)
- Carefully pour 5-6 g of rock powder into the bottle (for samples with >1% TOC, less material is sufficietnt). Pouring is cleaner than using a spatula. The sample will not be weighed after the maceration (residue too small for accurate mass), so this weight does not need to be accurate. It is just recorded to keep samples consistent and to avoid an unbalanced load in the centrifuge.
- If powder gets stuck on the outside of the thread of the bottle, carefully blow it off with air or wipe it off with a kimwipe. Otherwise, the bottle may not seal well
- Wrap ~20cm of Teflon tape around the thread. This will improve the seal. It needs to be redone each time the bottle is used for a new sample. If leakage is observed later during the process, wrap another piece of Teflon tape over the first one.
Optional: remove excess carbonate with HCl
- If samples are very carbonate-rich, then it is better to remove that with HCl before adding HF, because excess Ca2+ ions may enhance the formation of insoluble fluoride precipitates.
- Use the graduated cylinder to add 50ml of water and then 50ml of concentrated HCl to the sample.
- Add the acid carefully and gently stir the sample during the process to avoid overflow.
- Wait until the fizzing has subsided before you tighten the cap of the bottle
- Label the cap and the bottle with the sample name. Some bottles and caps have Roman numerals scratched into them that can also be recorded for better identification.
- Cover each cap with a piece of Al foil (~10x10cm). This is useful, because it serves as an indicator of acid escape from the inside of the bottle. Al reacts with acid.
- Place the capped bottles into the shaking water bath at 55°C, 150 rpm overnight. At higher speeds, the water bath tends to stop after several minutes for unknown reasons. But this speed works well enough. Make sure the water is filled up to about ½ the height of the bottles. If it is too full, it may spill.
- The next day, first check if bottles leaked, i.e. if the Al foil appears corroded. Discoloration to brown is a normal reaction to hot DI water and not a corrosion effect. Corrosion makes the foil crumble and turn white. If leakage is suspected, carefully remove all bottles from the water bath, neutralize the water in the bath with baking soda and replace it (see below)
- If any of the bottles have lost significant amounts of volume, top them up before centrifugation to keep bottles balanced. Use Teflon tape to seal the caps better.
- Place the bottles into the centrifuge, using the large green rotor. Make sure the rotor is selected from the menu (compare names written on the outside of the rotor). Balance the load.
- Centrifuge at 3000-3500g for 30min at room temperature. The maximum allowable speed for these bottles is 4000g, but given the long centrifugation time, that speed can actually cause breakage. At 3000g, the centrifuge may produce a message saying that the speed is low, but it will function as usual.
- Carefully decant the acid into a dedicated waste bottle.
- Add 200ml of DI-water, either with the graduated cylinder, or using the calibrated Teflon bottle where 100ml and 200ml are marked on the outside. Place the sample bottle next to the marked bottle, then fill the sample bottle up to that mark.
- Shake up the sample with the water by hand, then place the bottle back into the centrifuge (3000-3500g for 30min).
- Decant the water into the HCl waste. This step is for washing out the residual HCl.
Dissolution of silicates with HF
- If you performed the HCl step, then it is advisable to place another layer of Teflon tape around each step while the bottles are all empty.
- Add 100ml of DI-water, followed by 100ml of HF into the sample bottle. This can be done with either the graduated cylinder or with the marked Teflon bottle. The marked bottle gives more stability than the cylinder. Place your sample bottle next to it, then fill the sample bottle up to the mark.
- Cover the bottles with a fresh piece of Al foil as an indicator of acid leakage (see above for HCl)
- Place the bottles in the shaking water bath and shake at 55°C, 150 rpm overnight (see above)
- The next day, check for acid leakage manifested by corrosion in the Al (see above). In the event of leakage, neutralize the water in the bath with baking soda, then replace it. Top up the sample bottles that leaked.
- Place the sample bottles into the centrifuge (balanced) and centrifuge at 3000-3500g for 30min.
- Decant the HF into the HF waste. At this point, kerogen may be floating in the liquid and try to escape while pouring. You can use the Teflon spatula to hold it back as much as possible.
Dissolution of fluoride precipitates with BF3
- In most cases, the samples contain some amount of fluoride precipitates after treatment with HF. These precipitates are only weakly soluble in water, but can be dissolved in a mixture of boric acid and hydroflyuoric acid, which combined generate BF3 molecules.
- To make BF3, weigh out 62.5g (61-64g are ok) of H3BO3 per sample into a clean weigh boat. Boric acid usually comes as a powder, but sometimes the powder is clumped together. Use a Teflon spatula to break it up for easier weighing.
- Pour the powder into a clean 250ml Teflon bottle.
- Use the graduated cylinder to add 100ml of DI-water. The marked bottle can’t be used for this step, because the total volume of the mixture will exceed the 100ml mark.
- Use the graduated cylinder to add 100ml of concentrated HF.
- Cap the bottle and shake up the mixture (now BF3) until the powder is dissolved. This may take a few minutes. The dissolution is an exothermic reaction and will generate heat, but no effervescence.
- Pour 200ml of the BF3 into the sample bottle. There will be some residual BF3 that won’t fit and that can go into the HF waste bottle. Do not fill the sample bottle to more than 200ml, because that will enhance leakage during the centrifugation. Do not leave the residue in the BF3 bottle, because then the next load for the next sample won’t fit.
- Cap the bottles tightly and place it back into the shaking water bath at 55°C, 150 rpm overnight. Use Al foil to check for leakage as above.
- The next day, centrifuge the bottles at 40°C, 3000-3500g for 30min. The higher temperature helps keep boric acid in solution.
- Decant the BF3 into the HF + H3BO3 waste bottle. Use the Teflon spatula to minimize kerogen loss (see above).
- While the centrifuge is running, heat up 200ml of DI-water per sample in a clean glass beaker on the hotplate. Cover the beakers with Al foil during the heating to keep stuff from falling in.
- After decanting the BF3, rinse the sample three times with hot DI-water. Hot water is needed to keep boric acid in solution. It can work without heat, but this step is usually more successful with heated water. Flaky residues of boric acid can also be dissolved by placing the bottles with hot water into the sonic bath for 10-20min between centrifugations.
- After three washes (decanting the water into the HF-waste each time), carefully pour the residual liquid with the kerogen into a scintillation vial. If necessary, use 2 vials per sample to get all of the liquid out. Instead of pouring, you can use a disposable transfer pipette to transfer as much kerogen as possible to the scintillation vial. If the kerogen is sticky at the bottom of the Teflon bottle, place it into the sonicator for several minutes. There will always be residues of kerogen that cannot be retrieved.
- Label each vial, cap them, and place them into a quiet place for several hours until the kerogen has settled or is floating at the surface.
- Use a disposable transfer pipette (a new one for each sample) to remove as much of the clear water as possible from the vials. If more than one vial was used per sample, the aliquots can now be re-combined. This may require another round of settling and pipetting.
- Once the liquid level is down to less than 0.5cm, loosely cap the vials (tight capping can cause explosion in the freeze drier). Place each vial into a separate clean glass beaker and cover that with Al foil. This minimizes cross-contamination between samples and sample loss.
- Place the samples into the freezer overnight. Sometimes the kerogen prevents the water from freezing, but ideally, this step minimizes ‘boiling’ and spillage in the freeze drier.
- The next day, place the samples into the freeze-drier and dry them for 1-2 days until all ice has disappears.
- Now the dry kerogen should be ready for analysis.
Washing the Teflon bottles
- Do not use a brush to clean the bottles, because that will scratch the Teflon and make it even stickier next time.
- Carefully remove any Teflon tape without scraping the threads.
- Fill each bottle with soapy water and use a paper towel and your fingers to wipe it as much as possible on the inside. You can use a soft Teflon stick to get the paper towel down to the bottom
- Rinse out the soapy water with DI-water
- Fill the bottles with concentrated bleach (Chemstore or drugstore) and place them on a hotplate, inside a glass beaker or glass dish. Otherwise, the bleach may corrode the hotplate if it drips out. Leave the bottles on the hotplate at ~100°C overnight. This should oxidize all visible kerogen. If one night is not enough, leave the bottles on the hotplate until no more kerogen residues are visible. The caps may need to be boiled in bleach separately to remove all kerogen from the threads.
- Discard the bleach and thoroughly wash the bottles with DI-water.
- Pour ~10-20ml of alcohol into each bottle, cap them and put them back on the hotplate to let the alcohol reflux for several hours or overnight.
- Discard the alcohol, rinse the bottles thoroughly with DI-water.
- Pour 10-20ml of 6M HCl into each bottle, cap them and let the acid reflux for several hours or overnight on the hotplate.
- Discard the HCl and rinse the bottles thoroughly with DI-water.
- Place the bottles on the hotplate or into the drying oven to dry. Make sure that they are exposed for as little time as possible. Let the opening face backwards towards the wall to keep dust from flying in.
- To indicate that a bottle is washed and ready to use, place a piece of Al foil over each cap. This also keeps them clean.
- Removing water from the water bath: This is best done with a hose. Stick one end into the water, briefly suck on the other hand, then let the water run downhill into the dedicated glass beaker. Replace the water with tap DI-water. This is not 18 MΩ as in all other cases above, but clean enough for this purpose while not as corrosive.
- Rust in the water bath: Apply ‘anti-rust’ spray, especially at the arm that connects the shaking table to the motor. Keep the water bath empty when it is not being used. This will reduce the risk of rusting.
- Placing bottles into the water bath: For the 250ml Teflon bottles, strap one of the blue bottle racks to the shaking table in the bath. Use zip-ties for that. The alternative metal clamps are not sturdy enough for the bottles when they are full.
- Acid spills: use excess calcium carbonate to neutralize the acid. Follow safety procedures.
- Robl TL, Davis BH. (1993) Comparison of the HF-HCl and HF-BF3 maceration techniques and the chemistry of resultant organic concentrates. Organic Geochemistry 20, 2. doi: 10.1016/0146-6380(93)90042-A.
- Stüeken EE, Buick R, Guy BM, Koehler MC. (2015). Isotopic evidence for biological nitrogen fixation by molybdenum-nitrogenase from 3.2 Gyr. Nature 520. doi: 10.1038/nature14180.