Chromatography Flashcards
(34 cards)
Chromatography is also a form of what?
Packed bed adsorption
What is chromatography widely used for
High resolution purification of proteins
What 3 things can chromatography be used for that isn’t the high resolution purification of proteins
- Analysis
- Foods and neutraceuticals
- Diagnostics and monitoring
What is adsorption
The binding of molecules (adsorbate) to the surface of a solid (adsorbent)
What does adsorption involve?
The transfer of dissolved solutes from the liquid phase to the solid phase
What is adsorption influenced by? (4)
- Molecular weight, size and shape
- Shape of the binding site or ligand
- Polarity of molecule or adsorbent
- Electrostatic charge of molecule or adsorbent
What is the advantage of chromatography (2)
- High selectivity
- Ability to recover solutes from dilute solutions
What are the disadvantages of chromatography (4)
- 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
What are the key features of adsorbents
What can they be made from
What structure can they take
- High specific surface areas
- cellulose, silica, synthetic resins, agarose, dextran
- Packed bed, monoliths, membranes, fluidised beds
In adsorption, physical binding takes place due to one or more non-covalent bindings such as: (5)
- Van der waals
- Electrostatic interactions
- Hydrophobic interactions
- Hydrogen bonding
- Partitioning
What are 5 separation mechanisms
- Gel filtration
- Hydrophobic interaction
- Ion exchange
- Affinity
- Reversed phase
The performance of a chromatography column depends on
- Adsorption
- Binding capacity
- Resolution
- Efficiency
- Selectivity
What are 3 key isotherms used in chromatography
- Linear
- Freundlich
- Langmuir
To what situation does the linear isotherm apply to
Very low solute concentrations
What is the freundlich isotherm useful for?
Binding of antibiotics, hormones and steroids
Hydrophobic interactions follow the freundlich isotherm
When is the langmuir isotherm applicable?
When there is strong interaction between solute and adsorbent
What is the capacity factor
The capacity factor quantifies the extent to which a molecule interacts with the stationary phase
What is retention time?
The time between sample injection and an analyte peak reaching a detector at the end of the column
Define selectivity
The difference in interactions between two solutes with the stationary and mobile phases, and is independent of solute concentration
Describe the concept of a theoretical plate
A hypothetical zone in which an equilibrium established between 2 phases
The greater the number of plates the better the separation
What are assumptions of the plate model
- 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
Define the terms in the Van Deemter equation and explain how they can vary
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
What interactions are involved in Ion - exchange chromatography
Electrostatic
Describe the key concepts behind ion exchange chromatography (5)
- 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
