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Mirella Di Lorenzo > Chromatography > Flashcards

Chromatography Flashcards

1
Q

Chromatography is also a form of what?

A

Packed bed adsorption

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2
Q

What is chromatography widely used for

A

High resolution purification of proteins

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3
Q

What 3 things can chromatography be used for that isn’t the high resolution purification of proteins

A
  • Analysis
  • Foods and neutraceuticals
  • Diagnostics and monitoring
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4
Q

What is adsorption

A

The binding of molecules (adsorbate) to the surface of a solid (adsorbent)

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5
Q

What does adsorption involve?

A

The transfer of dissolved solutes from the liquid phase to the solid phase

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6
Q

What is adsorption influenced by? (4)

A
  • Molecular weight, size and shape
  • Shape of the binding site or ligand
  • Polarity of molecule or adsorbent
  • Electrostatic charge of molecule or adsorbent
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7
Q

What is the advantage of chromatography (2)

A
  • High selectivity

- Ability to recover solutes from dilute solutions

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8
Q

What are the disadvantages of chromatography (4)

A
  • Surface phenomenon (binding is restricted to the surface)
  • Can only be run in batch mode (cyclic process)
  • Can result in loss of protein activity
  • Adsorbents can be fouled irreversibly
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9
Q

What are the key features of adsorbents
What can they be made from
What structure can they take

A
  • High specific surface areas
  • cellulose, silica, synthetic resins, agarose, dextran
  • Packed bed, monoliths, membranes, fluidised beds
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10
Q

In adsorption, physical binding takes place due to one or more non-covalent bindings such as: (5)

A
  • Van der waals
  • Electrostatic interactions
  • Hydrophobic interactions
  • Hydrogen bonding
  • Partitioning
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11
Q

What are 5 separation mechanisms

A
  • Gel filtration
  • Hydrophobic interaction
  • Ion exchange
  • Affinity
  • Reversed phase
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12
Q

The performance of a chromatography column depends on

A
  • Adsorption
  • Binding capacity
  • Resolution
  • Efficiency
  • Selectivity
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13
Q

What are 3 key isotherms used in chromatography

A
  • Linear
  • Freundlich
  • Langmuir
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14
Q

To what situation does the linear isotherm apply to

A

Very low solute concentrations

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15
Q

What is the freundlich isotherm useful for?

A

Binding of antibiotics, hormones and steroids

Hydrophobic interactions follow the freundlich isotherm

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16
Q

When is the langmuir isotherm applicable?

A

When there is strong interaction between solute and adsorbent

17
Q

What is the capacity factor

A

The capacity factor quantifies the extent to which a molecule interacts with the stationary phase

18
Q

What is retention time?

A

The time between sample injection and an analyte peak reaching a detector at the end of the column

19
Q

Define selectivity

A

The difference in interactions between two solutes with the stationary and mobile phases, and is independent of solute concentration

20
Q

Describe the concept of a theoretical plate

A

A hypothetical zone in which an equilibrium established between 2 phases
The greater the number of plates the better the separation

21
Q

What are assumptions of the plate model

A
  • Large number of theoretical plates
  • Equilibrations between the stationary and mobile phase occur in these plates
  • The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next
22
Q

Define the terms in the Van Deemter equation and explain how they can vary

A

A - Arises from the multiple pathways in the packed column. Reduces with smaller particles, homogeneous packing

B - Longitudinal diffusion as the band moves along. Reduces by increasing flow rate and reducing particle size

L - Mass transfer of the solute in and out of the stationary phase. Reduces with smaller particles and greater prosity

23
Q

What interactions are involved in Ion - exchange chromatography

A

Electrostatic

24
Q

Describe the key concepts behind ion exchange chromatography (5)

A
  • There are electrostatic interactions between molecule and adsorbent
  • Proteins have a net surface charge
  • Adsorbents have attached charged groups on insoluble particles
  • binding depends on pH, ionic strength, buffer type
  • Proteins net charge is affected by pH
25
Q

In hydrophobic interaction chromatography what does it rely on

A

Interactions between hydrophobic patches on the protein surface and the adsorbent

26
Q

How is desorption achieved in hydrophobic interaction chromatography

A

Lowering salt concentration

27
Q

What is affinity chromatography based on

A

Biologically specific recognition of target molecules by ligands

28
Q

What is an obvious advantage of affinity chromatography

A

Very specific - can recover a solute from a complex mixture of hundreds of other solutes

29
Q

List some affinity ligands

A
  • Dye ligands
  • Antibodies
  • Enzyme substrate
  • Lectins
  • Concanavalin
  • Protein A
  • Immobilised metal ions
  • Glutathione
30
Q

How is a molecule desorbed from a ligand

A

By using conditions that do not favour the affinity interactions

  • extremes of pH
  • ionic strength
  • competitive ligand
  • high concs of urea an guanidine
31
Q

Be aware of the steps for the production of a fusion protein

A

Lecture 8

32
Q

Describe fusion protein purification

A

bind target protein with affinity tail
affinity tail binds with ligand
cleavage of tail with enzymatic or chemical methods

33
Q

Using a specific protease for enzymatic cleavage has 1 advantage and 2 disadvantages

A

Selective but expensive and pretease must then be removed

34
Q

Using chemical cleavage has 1 advantage and 2 disadvantages

A

simple and cheap
May cleave protein mid molecule
harsh reaction conditions

Mirella Di Lorenzo (4 decks)

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