explain chromatography
- Technique for separating mixture into individual components
- Mixture passed through a medium with 2 phases
○ Stationary phase
○ Mobile phase - The molecule get distributed between these phases
- Separates using molecules
○ Size
○ Charge
○ Hydrophobicity
- Affinity - Note:
○ Amino acids separated by charge
Proteins separated by charge, size, affinity
- Mixture passed through a medium with 2 phases
explain the partition coefficient (K)
- Partition coefficient (K): the ratio of concentrations of a compound in a mixture of 2 immiscible solvents at equilibrium
- K is defined as the conc. Of solute in the stationary phase divided by the conc. Of solute in the mobile phase
- As K increases, the solute will take longer to pass through
- Note: the extraction time is shorter when more of the solute is in the mobile phase then the stationary phase
what is the partition coefficient (K) formula
REFER TO ONENOTE
explain analytical chromatography
§ Analysis of complex mixtures
§ Sample volume smaller
§ Shorter columns work
Small scale- molecules are separated + identified but not isolated
explain preparative chromatography
§ Purification of 1 compound from a mixture
§ E.g. Recombinant insulin from bacteria
§ Sample volume larger
§ Longer columns are better
On a larger scale- molecule is isolated
explain the retention factor Rf
REFER TO ONENOTE
what are the 4 stages of column chromatography
- Equilibration with buffer
- Sample application elution
- elution
Regeneration of column
explain ion exchange chromatography
- Used for amino acids + proteins
- Separates compounds based on charge
- Column contains porous charged solid phase (resin) with charged molecules
- Pos. Charged resin attracts neg. Charged analytes
- Analytes can be extracted by changing pH or salt conc.
- Strength of acidity/basicity of the resin + no./unit volume determines the type + strength of binding
- Fully ionised groups bind strongly, weak acids or bases, whose charge depends on pH do not bind as strongly
Strength of binding will determine what resin to use
explain cation vs anion exchange chromatography
cation
- Neg. Charged, so pos. Charged will bind strongly to this
- If pH over 7- basic
anions
- Pos. Charged, so neg. Charged will bind strongly to this
- If pH under 7- acidic
explain pI
- Is the pH at which a particular molecules carries NO net electrical charge
- Also point were proteins do not mvoe in a electric field (in electrophoresis)
It depends on net. No. Of acidic/basic residues
- Also point were proteins do not mvoe in a electric field (in electrophoresis)
what factors are proteins seperated by, and what type of techniques can be used for it
- Seperated by
○ Charge
§ Ion exchange chromatography○ Size § Size exclusion chromatography or gel filtration ○ Hydrophobicity § Hydrophobic interaction chromatography or reverse phase chromatography ○ Affinity Affinity chromatography
explain gel filtration/SEC
- Separates molecules based on size
- Molecules portioned between mobile + stationary phase
- Made of a porous matrix
- Protein size + shape effect migration of proteins through the pores (large proteins elute before smaller proteins)
- Molecular weight ranges can be manipulated by bead/pore size
Isocratic elution: eluent not important as long as molecules are soluble
**what are the important parameters of gel filtration/SEC
○ External volume (void volume) Vo
○ Sum of external + interval volume = total volume, Vt
○ Elution volume, Ve
The partition coefficient in SEC is Kav
explain the Kav formula
REFER TO ONENOTE
explain the affinity chromatography
- Uses the specific binding properties of the protein
- Stationary phase is modified with a small biomolecule e.g. Ligand, covalently or chemically attached with specific affinities
○ Enzyme substrate
○ Enzyme inhibitor
○ Antibody
○ Nitrilotriacetic acid - Only the protein of interest will bind to the column
Elution with free ligand or by weakening the binding
- Stationary phase is modified with a small biomolecule e.g. Ligand, covalently or chemically attached with specific affinities
explain the affinity chromatography process
- The metabolite can be immobilised by covalently coupling it to an insoluble matrix (e.g. Agarose polymer)
§ Cell extracts containing many individual proteins may be passed through the matrix
2. A protein interacts with metabolite
§ The metabolite is thus a ligand that binds specifically to this protein
3. Purification of proteins
4. Adding an excess of free metabolite will compete for the bound protein, dissociates the protein from the chromatographic matrix
§ The protein passes out of the column complexed with free metabolite
5. Specific protein bind to ligand
All other unbound material is washed out of the matrix
explain the hydrophobic interaction chromatography
- Also called reverse phase chromatography
- Uses hydrophobic nature of proteins
- Hydrophobic groups are covalently linked to the solid phase matrix (e.g. Phenyl groups)
- In the presence of high salt concs. Proteins bind to the matrix through hydrophobic interactions
Proteins can be differentially eluted by lowering the salt conc. Or by adding solvents
explain electrophoresis
- The movement of ions through an electric field
- Proteins seperated by size
- SDS-PAGE is a type of electrophoresis
explain SDS-PAGE
- The hydrophobic tail of SDS strongly interacts with polypeptide chains
- SDS (a detergent) disrupts protein folding + adds heat and a agent to break disulphide bonds + unfold proteins, remove tertiary structure
- SDS is neg. Charged
- So applying electric field allows serrate of proteins by size in the migration through the porous gel
-uses a stain
