Lipid Analysis

Question 1

How do you separate lipids from other molecules

Answer 1

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

Question 2

Describe the solubility of lipids in water and organic solvent

Answer 2

soluble in organic solvent
insoluble in water

Question 3

How do you separate individual lipids?

Answer 3

Thin layer chromatography (TLC)

Question 4

describe TLC

Answer 4

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

Question 5

How can you visualize lipids on TLC plates

Answer 5

Non-destructive/ reversible: Iodine vapor, dichlorofluorescein
Destructive/ irreversible: Ninhydrin, Phosphoric acid

Question 6

Describe the sensitivity of the various visualization methods

Answer 6

Charring > iodine>dichlorofluorescein

Question 7

What is an Rf value

Answer 7

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

Question 8

What are the advantages and disadvantages of TLC

Answer 8

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)

Question 9

how can sensitivity of lipid analysis be improved?

Answer 9

Can be improved by using radioactive precursors, TLC plate scanner then measures the radioactivity (energy)

Question 10

T/F multiple solvent systems might be needed to separated lipids of interest.

Answer 10

true

Question 11

describe iodine vapor

Answer 11

reversible
relatively unspecific reagent and binds to double bonds

Question 12

Describe dichlorolfuorescein

Answer 12

reversible
General lipid stain, can view under UV light, binds to double bonds

Question 13

Describe Ninhydrin

Answer 13

Destructive/irreversible
Primary amine containing lipids
Heat 20 min at 120 C

Question 14

Describe phosphoric acid

Answer 14

Destructive/irreversible
Charring
unspecific
chars any organic material but lipids are the only material on the plate
Heat 5-30 minutes at 110 C

Question 15

limitations of Rf values

Answer 15

Some Rf values are similar, hard to know where to measure from on the plate

Question 16

Describe gas chromatography

Answer 16

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

Question 17

how can lipoproteins be separated

Answer 17

Ultracentrifugation
Separated by density

Question 18

Size-exclusion chromatography

Answer 18

Larger molecules move through the column faster

Question 19

Low and high tech alternatives to TLC limitations

Answer 19

Low tech: Two solvents, one half each
High-tech: 2-dimensions

Question 20

What level of LDL and VLDL is healthy and bad?

Answer 20

Healthy
LDL= <1.8 mmol/L
VLDL= <150 mmol/L
Bad
LDL= >5.2mmol/L
VLDL= >5.7mmol/L