Chapter 24 - High-Performance Liquid Chromatography Flashcards

0
Q

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

A

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

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1
Q

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

A

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.

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2
Q

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

A

At a low pH ligands are cleaved.

At a high pH the silica will dissolve.

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3
Q

What are some characteristics of normal phase chromatography?

A
  • 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
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4
Q

What are some characteristics of reversed phase chromatography?

A
  • 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
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5
Q

What is eluent strength?

A

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

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6
Q

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

A
  • used for protein purification.
  • proteins adsorbed when aqueous mobile phase contains high salt concentration.
  • elution strength is increased by decreasing salt concentration.
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7
Q

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

A
  • 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.
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8
Q

What is desalting?

A

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

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