Lipid Analysis Flashcards

1
Q

How do you separate lipids from other molecules

A

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

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2
Q

Describe the solubility of lipids in water and organic solvent

A

soluble in organic solvent
insoluble in water

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3
Q

How do you separate individual lipids?

A

Thin layer chromatography (TLC)

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4
Q

describe TLC

A

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

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5
Q

How can you visualize lipids on TLC plates

A

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

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6
Q

Describe the sensitivity of the various visualization methods

A

Charring > iodine>dichlorofluorescein

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7
Q

What is an Rf value

A

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

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8
Q

What are the advantages and disadvantages of TLC

A

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)

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9
Q

how can sensitivity of lipid analysis be improved?

A

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

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10
Q

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

A

true

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11
Q

describe iodine vapor

A

reversible
relatively unspecific reagent and binds to double bonds

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12
Q

Describe dichlorolfuorescein

A

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

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13
Q

Describe Ninhydrin

A

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

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14
Q

Describe phosphoric acid

A

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

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15
Q

limitations of Rf values

A

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

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16
Q

Describe gas chromatography

A

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

17
Q

how can lipoproteins be separated

A

Ultracentrifugation
Separated by density

18
Q

Size-exclusion chromatography

A

Larger molecules move through the column faster

19
Q

Low and high tech alternatives to TLC limitations

A

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

20
Q

What level of LDL and VLDL is healthy and bad?

A

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