Module 12: Chromatography

Question 1

Principle of chromatography

Answer 1

Separation of compounds based upon interaction with two phases (stationary and mobile)

Question 2

Peak area

Answer 2

Quantity detected

Used to measure concentration

Question 3

Clinical uses of chromatography

Answer 3

Toxicology, amino acids, glycohemoglobin

Question 4

Types of separation

Answer 4

Ion exchange (anion and cation)
Steric exclusion
Adsorption
Partition
Affinity

Question 5

Anion exchange chromatography

Answer 5

Stationary phase is positive

Selects anions from the sample

Question 6

Cation exchange chromatography

Answer 6

Stationary phase is negative

Selects cations from the sample

Question 7

Steric exclusion chromatography

Answer 7

Gel filtration, gel permeation

Stationary phase contains small pores that allow separation based on size and shape

Question 8

Adsorption chromatography

Answer 8

Solid phase has adsorptive sites (sticky) to capture compounds

Question 9

Partition chromatography

Answer 9

Separation based on polarity

Often used in HPLC and GC (GLC) systems

Question 10

Normal phase in partition chromatography

Answer 10

Stationary = polar
Mobile = non-polar

Question 11

Reverse phase in partition chromatography

Answer 11

More common than normal phase
Stationary = non-polar
Mobile = polar

Question 12

Affinity chromatography

Answer 12

Uses specific binding proteins (e.g. antibodies) to capture analyte

Question 13

3 chromatography methods

Answer 13

Paper/thin layer chromatography (TLC)
Gas liquid chromatography (GLC)
High performance liquid chromatography

Question 14

Paper/thin layer chromatography (TLC) stationary and mobile phases

Answer 14

Stationary: paper, can be mounted on glass or plastic support
Mobile: liquid solvent

Question 15

Paper/thin layer chromatography (TLC) clinical uses

Answer 15

Drug screens, amino acid screens

Question 16

Racing factor/retention factor

Answer 16

Distance compound moves/total solvent from distance

Used to identify compounds

Question 17

Gas liquid chromatography (GLC) stationary and mobile phases

Answer 17

Stationary: porous inert solid coated with liquid
Mobile: inert gas

Question 18

Gas liquid chromatography (GLC) clinical uses

Answer 18

Alcohol analysis (sample compounds must be volatile)
Can be coupled with a mass spectrometer for further identification of compounds

Question 19

High performance liquid chromatography stationary and mobile phases

Answer 19

Stationary: solid, or liquid coated in solid
Mobile: liquid

Question 20

High performance liquid chromatography clinical uses

Answer 20

Drugs and glycohemoglobins

Question 21

Retention time

Answer 21

Time from sample injection to peak height in detector

Question 22

Factors affecting retention time

Answer 22

Sample composition
Column temperature (hotter=faster)
Column packing (stationary phase material and amount)
Flow rate
Column length

Question 23

Chromatography resolution

Answer 23

Relative separation of two different peaks

Question 24

Factors affecting chromatography resolution

Answer 24

Column length
Column temperature
Stationary and mobile phase composition

Question 25

Column efficiency

Answer 25

Overall performance based on column packing

Question 26

Internal standard

Answer 26

Structurally similar reference compound of known concentration added to all samples to account for system variations

Question 27

Higher column temperature speeds up or slows down chromatography retention time?

Answer 27

Speeds up

Question 28

How is chromatography resolution affected by column length?

Answer 28

Longer is better

Question 29

How is chromatography resolution affected by column temperature?

Answer 29

Lower is better