Lipid separation methods

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khaengel
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265705
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Lipid separation methods
Updated:
2014-03-11 13:36:25
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MCDB chapter 10
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chapter 10
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  1. Lipid extraction in organic solvents:
    • Neutral lipids: ethyl ether, chloroform, or benzene.
    • Membrane lipids: polar organic solvents (ethanol or methanol), solvents reduce weak interactions.
    • Method: phase extraction using chloroform:methanol:water. this is one phase and extracts all lipids. Add more water and it separates into two phases: water/methanol phase on top with proteins and sugars, organic phase on the bottom with lipids.
  2. Adsorption chromatography:
    • Separate lipids based on polarity.
    • Column chromatography:
    • 1. column is packed with insoluable polar material (silica).
    • 2. Sample is added, polar lipids will bind to the column, neutral lipids do not bind to the column.
    • 3. column washed with solvents of increasing polarity, neutral lipids are eluted in the first wash (chloroform), other lipids are eluted using solbents of increasing polarity, very polar or charged lipids are eluted with alcohol.
    • Note: this is separation, not identification.
  3. Thin layer chromatography:
    • Separate lipids based on polarity.
    • Same principle as column chromatography except: a plate of glass is coated with silica, sample is amplified as a spot or line to the bottom of the plate, bottom of plate (up to the spot) is placed into solvent.
    • The solvent rises into the silica through capillary action - lipids separate.
    • The lower the lipid polarity, the more the lipid moves - does not bind as tightly.
    • Lipids are detected with iodine vaports, fluorescent molecules, or specific dyes.
    • How to tell what lipids are on the plate is to run a plate in the same solvents with known lipids and compare the two.
  4. Gas-liquid chromatography:
    • Separetes charged lipids best. This separates lipids based on- solubility and┬ávolatility in solvent.
    • Volatility in solvent: goes from liquid to gas state with increased temperature, many lipids are naturally volatile, those that are not volatile must be derivatized in order to separate them.
    • Method:
    • 1. Add sample (or derivatized sample) into column packed with inert material.
    • 2. Heat the column to volatilize the lipids to be separated.
    • 3. Force the volatilized lipids through the column using an inert gas (a noble gas, such as helium).
    • 4. Order of elution depends on the nature of the solid column support and the boiling point of the lipid (or derivative).
    • The first one to come out us the shortest to the longest, saturated to the unsaturated.

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