Lipid Analysis

Question 1

What are the general steps to isolating lipids from tissue?

Answer 1

1. add buffer with biological pH to sample
2. undergo physical disruption
3. use cholroform and methanol to separate lipids from other cell fractions

Question 2

Describe thin layer chromatography.

Answer 2

to identify lipids in sample-samples are put on glass plate with silica is stationary phase and solvent (diff depending on what lipids you want to see) is added and lipids move to capillary action

Question 3

What are some common solvents used in TLC?

Answer 3

chloroform, benzene, methanol and hexane

Question 4

What are the reversible methods of visualization on TLC plates?

Answer 4

Iodine vapor
Dichlorofluorescein

Question 5

How does iodine vapour work?

Answer 5

non specific binding agent to double bods

Question 6

How does dichlorofluorescin worK?

Answer 6

general lipid stain that binds to dbs and can visualize under UV

Question 7

What are the irreversible ways to visualize lipids on TLCs?

Answer 7

nihydrin and phosphoric acid?

Question 8

How does nihydrin work?

Answer 8

binds amine containing lipids, heat for 20mins at 120C stains purple

Question 9

How does phosphoric acid work?

Answer 9

chars any organic material, must heat 5-30mins at 110-more sensitive

Question 10

What is an Rf?

Answer 10

distance travelled by sample/distance travelled by solvent

Question 11

What are some drawbacks to using the Rf method?

Answer 11

lipids travel different distances in different solvents, and some can travel similar distances leading to misidentification

Question 12

What is a common solvent and ratio for measuring phospholipids?

Answer 12

chloroform: methanol:aluminum hydroxide (65:25:4)

Question 13

What are some advantages to using TLC?

Answer 13

can’t break down like computers and machines, reliable and easy, relatively inexpensive

Question 14

What are some cons to using TLC?

Answer 14

tedious, expensive for materials, not very sensitive, cannot separate all lipids on one plate and dangerous if you inhale solvents

Question 15

What is gas chromatography used for?

Answer 15

for separation and analysis of fatty acids

Question 16

How does gas chromatography work?

Answer 16

the samples are carried through an oven and carried through a circular column with an inert gas, detector records when it was passed through and how much

Question 17

What reaction must occur first before putting samples into a gas chromatographer?

Answer 17

methylation reaction-must use heat and low pH

Question 18

What is the main way of analyzing lipids in circulation?

Answer 18

Ultracentrifugation and high performance liquid chromatography

Question 19

How does ultracentrifugation work?

Answer 19

Separates the lipoproteins based on densities

Question 20

What are the drawbacks to using ultracentrifugation?

Answer 20

Takes a long time (12hrs) and uses specialized equipment for high speeds

Question 21

How does HPLC work?

Answer 21

The lipoproteins are separated based on size as the beads present in the tube trap the smaller lipoproteins (HDL and VLDL) and the longer ones elute first