Chapter 24 - High-Performance Liquid Chromatography

Question 0

How do you prevent a siloxane bond from hydrolyzing below a pH of 2?

Answer 0

The surface has to be modified with bulky isobutyl groups so that the siloxane bonds are protected from hydrolysis at a low pH

Question 1

How does the shape and size of silica particles affect liquid chromatography?

Answer 1

The more uniformly spherical that the silica particles are the more that you can pack into a column and the smaller the particles are in turn creates a higher back pressure.

Question 2

In silica based chromatography, what happens when you have a low pH and high pH?

Answer 2

At a low pH ligands are cleaved.

At a high pH the silica will dissolve.

Question 3

What are some characteristics of normal phase chromatography?

Answer 3

• polar stationary phase
• less polar solvent (non aqueous)
• more polar solvent has a higher eluent strength
• peptides may be eluted by increasing the polarity of the solvent

Question 4

What are some characteristics of reversed phase chromatography?

Answer 4

• non polar stationary phase
• polar solvent
• less polar solvent has a higher eluent strength
• tailing is eliminated
• less sensitive to the polar impurities in the eluent
• peptides may be eluted by decreasing the % water of the solvent

Question 5

What is eluent strength?

Answer 5

Eluent strength is the measure of a solvents adsorption energy on a given adsorbent.

Question 6

Another type of adsorption chromatography is hydrophobic interaction chromatography (HIC), what are the characteristics of this type?

Answer 6

• used for protein purification.
• proteins adsorbed when aqueous mobile phase contains high salt concentration.
• elution strength is increased by decreasing salt concentration.

Question 7

Another type of adsorption chromatography is hydrophilic interaction chromatography (HILIC), what are some characteristics of this type?

Answer 7

• useful for small molecules that are too polar to be retained by reversed phase chromatography.
• stationary phases are strongly polar.
• solutes equilibrates between the mobile phase and a layer of aqueous phase on the surface of the stationary phase.
• eluent strength is increased by increasing the fraction of water in the mobile phase.
• differs from that of normal phase chromatography in that water may be present.

Question 8

What is desalting?

Answer 8

Is when salts of low molecular weight can be removed from large molecules.
(Useful for changing the buffer composition of a macromolecule solution)