HPLC

Question 1

3 types of liquid chromatography methods:

Answer 1

paper chromatography
thin layer chromatography
liquid column chromatography

Question 2

Differences in preparative vs analytical LC?

Answer 2

Analytical:

• goal is to identify/quantify target molecules
• small volume/column size/particle size
• high pressure (HPLC or UHPLC)

Preparative:

• goal is to purify/collect (clear interferences)
• large volume/column/particle size
• low pressure

Question 3

What is the usual column/particle size and sample volumne for analytical LC?

Answer 3

Sample: 0.01 - several microL

column: mm

particle size: um

Question 4

what is the goal for preparative LC?

Answer 4

Purify/collect large quantity of molecule of interest (get rid of interferences)

Question 5

What is the usual column/particle size and sample volume for prep LC?

Answer 5

Volume: mL - L

column: 5-10cm
particle: >10um

Question 6

What is the pressure range for low pressure LC, HPLC, and UHPLC?

Answer 6

low pressure: <10 bars
HPLC: >50 bars
UHPLC: >600 bars

Question 7

the pressure reading is 450 bars. This is considered ____ LC.

Answer 7

HPLC

Question 8

the main parts of a HPLC system: (7)

Answer 8

Solvent(s)
Pump(s)
Mixer
Injector (autosampler)
Column
Detector
Waste

Question 9

What is the stationary phase in HPLC? What is the mobile phase?

Answer 9

stationary: column (packing material)
Mobile: solvent(s)

Question 10

What is responsible for maintaining the pressure and flow in the HPLC system?

Answer 10

pump

Question 11

Describe the path of the solvent through the system:

Answer 11

bottles -> tubing -> pump -> mixer -> (sample injected) -> column -> detector -> waste

Question 12

Types of LC columns: (6)

Answer 12

reverse phase
normal phase
ion exchange
size-exclusion
affinity
chiral

Question 13

What is reverse phase LC? give an example of a column and mobile phase.

Answer 13

nonpolar stationary phase + polar mobile phase

column: C18
solvent: aqueous (water/acetone/methanol)

Question 14

What is normal phase LC? give an example of a column and mobile phase.

Answer 14

polar stationary phase + nonpolar mobile phase

column: silica
solvent: organic (hexane)

Question 15

Advantage/disadvantage of smaller particle size in HPLC:

Answer 15

good: small particle -> small theoretical plate height (H); broader range of optimal flow rates
bad: higher back pressure

Question 16

parts of an LC pump: (6)

Answer 16

vacuum chamber
proportioning valve
inlet valve
outlet valve
damper
purge valve

Question 17

the y axis and x axis of a chromatogram:

Answer 17

y axis = detector signal

x axis = time

Question 18

parameters of a chromatographic peak:

Answer 18

peak height
peak width
peak area

Question 19

What is 2D-LC?

Answer 19

coupling 2 LC columns with different separating powers

further separate individual peaks - display results as 2D chromatogram