Liquid Chromatography

Question 1

Who first discovered Chromatography?

Answer 1

M. Tswett
* using CaCO3 and ethanol

Question 2

What are the TWO main phases of Chromatography?

Answer 2

1. Normal Phase
2. Reverse Phase

Question 3

What is the main principle of Chromatography?

Answer 3

All forms rely on analyte partitioning between the MOBILE and STATIONARY phases

Question 4

What is the equation for the Distribution Coefficient?

Answer 4

Kd = [C]s / [C]m

Question 5

What is the equation for the Partition Coefficient?

Answer 5

K = ms / mm

Question 6

What does Chromatography rely on?

Answer 6

Differences in K for different molecules

Question 7

What does Chromatography measure?

Answer 7

Compounds retention time

Question 8

What is the equation for Retention Time?

Answer 8

Question 9

What is the equation for Resolution?

Answer 9

Question 10

What are the THREE Separation Performance parameters?

Answer 10

1. SELECTIVITY - ability to discriminate related compounds
2. EFFICIENCY - a measure of diffusion effects
3. CAPACITY - amount of material that can be resolved

Question 11

What does Low Flow Rate indicate?

Answer 11

Increased diffusion

Question 12

What does High Flow Rate indicate?

Answer 12

Non-equilibrium partitioning

Question 13

What is the equation for Resolution and the parameters?

Answer 13

Question 14

What is the Isocratic elution mode?

Answer 14

Same eluent

Question 15

What is the Gradient elution mode?

Answer 15

Question 16

What phase is associated with Dipole-Dipole interactions during Separation / Retention?

Answer 16

Normal phase
* Al2O3, SiO2
* Apolar

Question 17

What phase is associated with Hydrophobic interactions during Separation / Retention?

Answer 17

Reverse phase
* Med polar to polar

Question 18

What is associated with Ionic interactions during Separation / Retention?

Answer 18

Ionic exchange
* High polarity

Question 19

What size beads are used in LC?

Answer 19

200-50 μm

Question 20

What size beads are used in FPLC?

Answer 20

30-50 μm

Question 21

What size beads are used in HPLC?

Answer 21

3-5 μm

Question 22

Put the following in order of lowest to highest pressure used
FPLC
HPLC
LC

Answer 22

LC, FPLC, HPLC

Question 23

What separation media is used in Normal Phase?

Answer 23

• SiO2
• Al2O3

Question 24

What separation media is used in Bonded Phase?

Answer 24

C18
*Octadecyl Silyl

Question 25

What FIVE components make up a HPLC set up?

Answer 25

1. Solvent 2. Pump 3. Injector 4. Column 5. Detector

Question 26

What is the most common detection option in Chromatography?

Answer 26

Photometric UV-Vis

Question 27

What detection option is used for non-absorbing molecules in Chromatography?

Answer 27

Refractometric (RI)

Question 28

What detection option is used for proteins in Chromatography?

Answer 28

Fluorescent

Question 29

What column is used for peptides /oligos?

Answer 29

C18 or C8 RP

Question 30

What buffer and pH is used for Ion-Pairing Mobile Phase of peptides /oligos?

Answer 30

* TEAA buffer * pH 6

Question 31

What gradient is used for peptides /oligos?

Answer 31

Acetonitrile 0 - 100%

Question 32

What detection is used for peptides /oligos?

Answer 32

UV at 260 nm