Conversion of Fatty Acid Methyl Esters by Saponification 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. Prepare a solution of 2 N KOH in 5% H₂O in methanol.
   1. KOH and methanol can be purchased from chemical retailers. These chemicals should be pure and free of hydrocarbons.
      1. The molar mass of KOH is ~56 amu yielding 1 mol of OH^- for each mol of KOH. Thus, to achieve a 2 N concentration, dissolve 112 g of KOH in 0.95 L of pure water and 0.05 L of methanol.
      2. Note that the dissolution of KOH in water is exothermic and creates a large amount of heat. Take care to add the KOH pellets to the water slowly to avoid a violent reaction.
   2. Dissolve 112 g of KOH pellets into 0.95 L of pure water on an automatic stir plate.
   3. Add 0.05 L of methanol once all of the KOH is dissolved to aid in the dissolution of the organic biomarkers into the aqueous solution.
3. Prepare a solution of 6 N hydrochloric acid (HCl) in H₂O.
   1. HCl can be purchased from chemical retailers. This chemical should be pure and free of hydrocarbons.
   2. HCl generally comes concentrated as 13 N. Thus, a 1:1 mixture of HCl and pure water produces a 6.5 N mixture of HCl, which is close enough to 6 N for the purposes of this experiment.
      1. Be sure to add the HCl to the water and not the other way around, as adding water to concentrated HCl is exothermic and generates heat. This may cause HCl to splash.
   3. Gently and slowly pour 100 mL HCl into a beaker containing 100 mL pure water,swirling the mixture between additions of HCl.
4. Prepare a solution of 5% NaCl (table salt) in H₂O.
   1. NaCl can be purchased from chemical retailers. This chemical should be pure and free of hydrocarbons.
   2. Calculate and weigh the mass of salt needed to make ~1 L of 5% solution (w/w). 1 L of water weighs ~1 kg or 1,000 g. Thus, 50 g of NaCl dissolved into 950 mL gives a 5% solution (50 g+ 950 g = 1,000 g; 50 g / 1,000 g = 0.05 or 5%).
   3. Gently add 50 g of NaCl to pure water on an automatic stir plate and wait for it to dissolve.
5. Obtain the following materials: 2 clean and combusted (550 ˚C for 6 h) 40 mL borosilicate glass vials with PTFE-lined cap; a warming oven or heating blocks; combusted (550 ˚C for 6 h) borosilicate glass pipettes and bulbs; pH tape (acidic range); Hexane (hexane can be purchased from chemical retailers. This chemical should be Optima Grade or equivalent).

2. Methods

1. Start with the dried TLE (containing esters) in one of the 40 mL borosilicate glass vials.
2. Add approximately 10 mL of 2 N KOH to the TLE and cap.
3. Heat in the oven or on a heating block to 60 ˚C for 2.5 h to cleave the ester bond (Figure 3).
4. Remove the sample from heat and allow it to cool to room temperature.
5. Add approximately 10 mL of the 5% NaCl solution to the TLE (Figure 3). This helps keep the interface between the aqueous and organic phase (to be added) from foaming. Cap and shake briefly.
6. Add 6 N HCl to the salted TLE dropwise until pH 2 is reached to protonate the O^- and form the final product, a stable carboxylic acid (Figure 3; use pH paper to test). If the TLE was colored,there may be a shift in color coinciding with pH 2.
7. Add approximately 20 mL of hexane to the acidified solution, which is now ester-free. Cap and shake vigorously for 5 s to extract organic compounds from the water.
8. Allow to set until aqueous and organic phases completely separate. Salts, ions, water, and unreacted HCl remain in the aqueous phase. Organic compounds are now in the hexane.
9. Remove approximately 75% of the supernatant hexane using a pipette and dispense into the other 40 mL borosilicate glass vial.
10. Repeat 2.7 - 2.9 twice, adding approximately 10 mL of hexane each time.
11. Discard leftover aqueous mixture in a proper waste container.
12. Label the vial containing hexane and ester-free organics "TLE - saponified.