Lipid Analysis Flashcards
How do you separate lipids from other molecules
Combine tissue with biological buffer –> physical disruption –> mix lysate with organic solvent
Lipids will dissolve in the organic solvent, other components will be in aqueous phase
Describe the solubility of lipids in water and organic solvent
soluble in organic solvent
insoluble in water
How do you separate individual lipids?
Thin layer chromatography (TLC)
describe TLC
Lipid samples applied to a TLC plate: Glass plate with a thin layer of silica (stationary phase)
Placed in an organic solvent (mobile phase)
Non-polar molecules will move up faster and further on the plate
How can you visualize lipids on TLC plates
Non-destructive/ reversible: Iodine vapor, dichlorofluorescein
Destructive/ irreversible: Ninhydrin, Phosphoric acid
Describe the sensitivity of the various visualization methods
Charring > iodine>dichlorofluorescein
What is an Rf value
Retention factor, allows you to identify unknown lipids,
Distance traveled by sample/ distance traveled by solvent
greater Rf = more non-polar
can use Rf values to identify lipids
What are the advantages and disadvantages of TLC
Advantages: no moving parts (cant break down), Easy and reliable, relatively inexpensive
Disadvantages: Tedious (boring and can lose track), expensive, Can’t separate all lipids on one plate, not healthy (inhalation of solvents), not very sensitive (can be improved)
how can sensitivity of lipid analysis be improved?
Can be improved by using radioactive precursors, TLC plate scanner then measures the radioactivity (energy)
T/F multiple solvent systems might be needed to separated lipids of interest.
true
describe iodine vapor
reversible
relatively unspecific reagent and binds to double bonds
Describe dichlorolfuorescein
reversible
General lipid stain, can view under UV light, binds to double bonds
Describe Ninhydrin
Destructive/irreversible
Primary amine containing lipids
Heat 20 min at 120 C
Describe phosphoric acid
Destructive/irreversible
Charring
unspecific
chars any organic material but lipids are the only material on the plate
Heat 5-30 minutes at 110 C
limitations of Rf values
Some Rf values are similar, hard to know where to measure from on the plate
Describe gas chromatography
Separates fatty acid derivatives (methyl esters) (trans-esterification)
heat from oven makes sample volatile which is carried through column by gases
short chain fatty acids are eluted first
how can lipoproteins be separated
Ultracentrifugation
Separated by density
Size-exclusion chromatography
Larger molecules move through the column faster
Low and high tech alternatives to TLC limitations
Low tech: Two solvents, one half each
High-tech: 2-dimensions
What level of LDL and VLDL is healthy and bad?
Healthy
LDL= <1.8 mmol/L
VLDL= <150 mmol/L
Bad
LDL= >5.2mmol/L
VLDL= >5.7mmol/L
