Removal of Branched and Cyclic Compounds by Urea Adduction for U^k'₃₇ Paleothermometry

1. Setup and Preparation of Materials

1. Obtain a total lipid extract (TLE) using a solvent extraction method (Sonication, Soxhlet, or Accelerated Solvent Extraction (ASE)).
2. Purchase the following materials from any chemical retailer: combusted borosilicate glass pipettes and bulbs; pure water; hexane; Dichloromethane (DCM); methanol; urea.
   1. The reagents should be pure and free from hydrocarbons. Alternatively, pure water can be made in a lab using a water purification system.
3. Obtain 4-mL borosilicate glass vials with PTFE-lined caps.

2. Methods

1. Make a mixture of 2:1 DCM:Hexane (v:v).
   1. In an Erlenmeyer flask, mix 200 mL of DCM and 100 mL of hexane.
2. Make a mixture of urea in methanol (100 mg/mL).
   1. Pour 300 mL of methanol into an Erlenmeyer flask.
   2. Add 300 mg of urea.
   3. Stir on an automatic stir plate until the urea is completely dissolved.
3. Start with the dry sample in a 4-mL vial.
4. Suspend the sample in 1.5 mL of a 2:1 mixture of DCM:Hexane (v:v).
   1. If the sample does not completely dissolve, sonicate for 5 min.
5. To this mixture, add 1.5 mL of urea in methanol.
   1. After this addition, watch for a white precipitate to form. This signals the creation of urea crystals.
6. Gently dry the crystals under nitrogen, using gentle heat. Ensure all solvent has evaporated before proceeding to the next step.
7. Rinse the crystals 3x with approximately 1 mL of hexane. Remove the hexane between each rinse using a glass pipette to transfer it into a vial labeled "non-adduct."
8. Dissolve the crystals in 2 mL of pure water. Shake to ensure complete dissolution.
9. Extract the aqueous phase with hexane by adding 1 mL and gently shaking for 5 s.
10. Allow the hexane and water to completely separate before removing approximately 75% of the hexane using a glass pipette and transferring it into a vial labeled "adduct."
11. Repeat step 2.10 twice.
12. Dispose of the water and urea solution in an appropriate waste container.