Lipids – Extraction and Analysis by Thin Layer Chromatography and Gas Chromatography Haley Gale 3/12/13 Section: 02 Purpose: The purpose of this laboratory activity is to introduce us to the theory and application of chromatography, especially as they apply to thin layer chromatography and gas chromatography. More specifically 1.) Paper chromatography of black ink will be performed to illustrate the classic application of chromatography. 2.) Thin layer chromatography will be used to separate and identify the main lipid components of extracts prepared from plant vs. animal origins. 3.) Fatty acid methyl esters will be made out of fatty acid constituents of the same plant and animal lipid extracts to determine their fatty acid composition via gas chromatography 4.) The fatty acid composition of E. coli cells growing at 22 vs. 42 degrees Celsius will also be measured by gas chromatography to determine the effects of growth temperature fatty acid composition. Data: Table 1: Gas Chromatography Results, fatty acid detected and the percent composition. Fatty Acid Name & Symbol E. Coli 22°C Heptadecenoic Acid (17:1) Stearic Acid (18:0) Oleic Acid (18:1Δ9) Linoleic Acid (18:2Δ9,12) Linolenic (18:3Δ9,12,15) Lauric Acid (12:0) Myristic Acid (14:0) 3-Hydroxytetradecanoic Acid (3-OH-14:0) Myristoleic Acid (14:1) Palmitic Acid (16:0) Palmitoleic Acid (16:1) Margaric Acid (17:0) Nonadecanoic Acid (19:0) Eicosadecanoic Acid (20:2) Figure 1: Thin paper chromatography Percent Composition E. Coli - 42°C Beef Fat 1.25 29.46 1.86 12.09 6.05 4.2 1.43 30.99 20.5 4.32 1.84 2.08 8.24 5.895 1.88 40.44 11.275 8.59 6.68 1.07 2.33 17.51 44.96 2.02 Soybean Seed 1.99 34.99 23.79 30.43 2.29 25.50 3.52 1.96 8.83 Figure 2: Paper chromatography Questions: 3. The theory of gas chromatography is that the sample goes through difference phases to ultimately produce a retention time that can be used to identify a substance and its amount. The stationary phase (injecting the sample), the mobile phase (inert gas carrier), vaporization (rapid heating), and lastly detecting various resolved sample components. 5. The Rt is “retention time” which has different characteristics for each substance per say a “finger print.” The retention time is used to identify a substance in an unknown sample/mixture via gas chromatography. The Rf stands for “retention factor” which is relatively constant so can be used to identify and unknown when compared to migration standards. The retention factor is the distance a particular solute component migrates relative to the distance that the developing solvent front moves in a given chromatography system. 6. In animals lipids are often associated with lipoproteins of blood serum that are involved in the transport of lipids throughout the animal body. In plants a variety of acyl lipids are found on their external surfaces to form the cuticular layer that provides a waxy waterproof barrier that prevents tissue desiccation. 7. Although they are both chemically composed of “triacylglycerols”, they differ in that animal fats are characteristically enriched in the saturated fatty acids (16:0 – 18:0) which plant oils are enriched in the polyunsaturated fats (18:2 – 18:3). Bacteria contain fatty acids (mono- branched) may have also a methoxy- or a hydroxyl- substitution. I suppose that there is typically a preponderance of fatty acids with even numbers of carbon atoms in plants and animals because they have to maintain optimum membrane fluidity. Membrane fluidity is crucial in living organisms especially those that cannot regulate their body temperatures. 8. The main difference in the fatty acid composition of E. coli cells grown at 22 versus 42 degrees Celsius is that grown at 22 C ̊ it contains less fatty acid that at 42 ̊C which is because the optimal growth for E. coli is at 37 ̊C which is closer to 42 ̊C than 22 ̊C. 9. The main difference in the fatty acid compositions of soybean seeds versus beef fat is that soybeans contain more fatty acids that beef fat. The main difference in consuming animal based fats versus plant based fats is that animal fats (saturated fatty acids) can essentially solidify in undesirable locations in the human body such as the walls of coronary arteries. Such saturated fatty acids are thus thought to contribute to cardiovascular disease in humans. Conclusion: In conclusion we were able to apply three versions of chromatography to this lab. The fatty acid composition of an organism increases as it is incubated closer to its optimum growing temperature. Beef fat/animal fat in general contains less fatty acid by percent composition via gas chromatography than soybeans. Animal based fats also even though they contain less fatty acid are generally more harmful heath wise due to their ability to solidify.