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Overview

1:03

Principles of Saponification

2:38

Hydrolyzing Fatty Acid Methyl Esters

4:00

Separating the Organic and Aqueous Phases

5:36

Applications

8:03

Summary

Conversion of Fatty Acid Methyl Esters by Saponification for Uk'37 Paleothermometry

Source: Laboratory of Jeff Salacup - University of Massachusetts Amherst

The product of an organic solvent extraction, a total lipid extract (TLE), is often a complex mixture of hundreds, if not thousands, of different compounds. The researcher is often only interested in a handful of compounds or, if interested in many, may need to remove unwanted constituents that are"in the way" or co-eluting. For example, the concentrations of individual compounds in a sample are often determined on a gas chromatograph coupled to a flame-ionizing detector (GC-FID), because the relationship between FID response (in pA) and the amount of compound in a sample (e.g., ng/µL) is both linear and sensitive. The GC portion of the instrument separates different compounds in a sample based on their boiling point, chemical structure, and affinity with a solid phase that can change according to application. The result is a chromatogram (Figure 1), showing the separation of different chemical constituents in time, as well as their relative concentration (calculated as the area under the curve). However, sometimes more than one compound elutes off the GC at a time (Figure 1). In this case, sample purification is required before compounds can be confidently quantified.
 

Figure 1
Figure 1. A chromatogram showing the separation of different chemical constituents over time and their relative concentration (area under the curve). Co-eluting and separated peaks are shown.

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 achi

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This purification produces a TLE free of esters that may be co-eluting with the alkenones. However, the purification produced carboxylic acids, which cannot be injected onto instruments commonly used to analyze samples for alkenone concentrations because of their low volatility. For example, the boiling point of hexane, a 6-carbon hydrocarbon, is 68 ˚C, but the boiling point of its acid (hexanoic acid) is 205 ˚C. Most GC amenable biomarkers have from 20 to 35 carbon atoms (boiling point generally increases with an increasing number

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As mentioned previously, saponification is commonly used in organic geochemistry labs to remove fatty acid methyl esters (FAMEs) of alkenones, called alkenoates, which co-elute with alkenones on gas chromatographs (Figure 1). Saponification is also used to "free" fatty acids "bound" to sediment or macromolecules.The degradation and preservation of organic matter and biomarkers in sediments involves the removal of functional groups (N, O, and S) and the eventual polymerization

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