Chromatography Flashcards
Types of chromatography
- Gel Filtration
- Ion Exchange Chromatography
- Affinity Chromatography
Properties of Proteins - MCHDM
- Mass
- Charge – pH
- Hydrophobic/Hydrophilic Properties
- Differential Solubility
- Mobility in Applied Fields
Hydrodynamic Properties of Proteins
(Aside)
Rh – hydrodynamic radius
K-1Debye Length
Y(r) – electrostatic potential
z- zeta potential
Techniques in Chromatography
- Gel filtration,
- Hydrophobic Interactions,
- Ion Exchange, Affinity,
- Reverse phase
Gel – Definitions
Gel is a semi-rigid mass of lyophilic sol in which the solvent has penetrated the sol particles
- Colloid – dispersion of small particles of one material in another
- Small ~ 500 nm
- Sol – Solid dispersed in a liquid
- Aerosol – liquid in a gas
- Emulsion – liquid in a liquid
- Smoke – solid in a gas
- Lyophilic colloid – solvent attracting colloid
- Lyophobic colloid – solvent repelling
Principles of Gel Filtration
Use of Gel Filtration
- To fractionate proteins of different MW’s
- To remove aggregated protein from purified sample to produce homogeneous protein sample
- To desalt protein
Choice of Gel
- Sephadex-Cross-linked polysaccharide (dextran)
- Usually as a bead
- Used to separate low and high molecular weights
- Desalting alternative to dialysis
- Buffer exchange removal of small molecules
Choice of Gel (resin)
- Sephacryl- Composite gel. Cross linked allyldextran with N,N’-methylene bisacrylamide- hydrophilic matrix of high mechanical strength.
- Sepharose- Bead formed gel prepared from agarose
- Combinations
- Sephadex will flow under gravity and often needs reduced operating pressure to prevent gel from packing down. Sephacryland Sepharosecan operate at higher pressures and often used on FPLC (Fast liquid Chromatography) system
SephacrylHigh Resolution (HR)
Sephacryl High Resolution (HR) is a composite gel prepared by covalently cross-linking allyldextran with N,N’-methylenebisacrylamideto form a hydrophilic matrix of high mechanical strength
Sepharose
- Sepharoseis a bead-formed gel prepared from agarose.
- In its natural state agarose occurs as part of the complex mixture of charged and neutral polysaccharides referred to as agar.
- The agarose used to make Sepharoseis obtained by a purification process which removes the charged polysaccharides to give a gel with only a very small number of residual charged groups.
- A gel forms spontaneously as a hot solution of agarose is cooled. The individual polysaccharide chains form double helices which subsequently aggregate to form bundles during the formation of a stable gel
Agarose Gel
Gel Filtration
- Molecules in solution are separated according to differences in their sizes as they pass through a column packed with a chromatographic medium which is a gel
Porous and Non-porous beads
Gel Filtration
- Relatively small molecules can diffuse into the gel from the surrounding solution and large molecules will be prevented from diffusing into the gel to some degree.
- Sufficiently large molecules are confined to the solution outside.
- Gel is packed into column in a continuous manner after degassing to remove trapped air.
- Parameters of column are important. Length is important since it affects both the resolution and time taken to elute it.
- Resolution - column length
- Elution time - column length
- Sample limited to 1-2% of column volume for best resolution
Diameter and Run-time
Separation of StefinB Oligomers
Column Length and Resolution
Excluded Volume
Determines, along with the MW, the residence time of the protein, when it will run, depends on the included and excluded volume of the column
- Tightly packed column
- Voutside< Vinside
- Generally, VO, is 1/3 of the column volume
- So Viis 2/3.
Protein Elution Time
Ion-Exchange Chromatography
- Separation based on charge on protein molecule.
- Proteins bind mainly by electrostatic forces between the proteins surface charges and dense clusters of charge groups on the ion-exchange resin
- Resin is a thick liquid that hardens to a transparent solid
- Proteins (analytes) are then retained on the column based on their charged (Coulomic) interactions.
- The counter ions in solution are exchanged for the ions on the resin
Elution of the Captured Protein
Continuous Elution Gradient
New Types of Ion Exchangers
- FPLC systems (Fast Protein Liquid Chromatography) runs columns at high pressure
- Mono Q and Mono S pre-packed columns (£500)
- FastflowQ and FastflowS materials
Hydrophobic Chromatography (HIC)
- Separation based on the hydrophobicity of the protein. Several hydrophobic column supports coupled to cross-linked agarose matrices.
- Protein is bound at high salt concentration- usually ammonium sulfate.
- It is eluted from the column by reducing the salt concentration with a linear gradient typically 1.5M ammonium sulfatein buffer to buffer with no salt
- Hydrophilic Interaction Chromatography
Proteins Separated by Hydrophobicity (HIC)
- HIC is related to reverse phase chromatography (RPC) techniques. Absorbents for RPC are more highly substituted with hydrophobic ligands than for HIC.
- HIC – 10-50 mM/mLgel of C2-C8 alkyl or simple aryl ligands
- RPC – Several hundred mM/mLgel of C4-C18 alkyl ligands.
- Proteins binding to RPC is very strong and non-polar solvents are needed for elution.
- RPC extensive applications in analytical and preparative separation of peptides that are stable in aqueous-organic solvents.
Affinity Chromatography
•Separation of proteins on the basis of a reversible interaction between a protein and a specific ligand coupled to a chromatographic matrix.
•Advantages
- High Selectivity
- High capacity for protein
- High purification of factor
•Antibody Capture
- Affinity confers
- Sensitivity
- Specificity
E. coli cell lysate HIC – RPC MS
