L8

Question 1

explain chromatography

Answer 1

• 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

Question 2

explain the partition coefficient (K)

Answer 2

• 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

Question 3

what is the partition coefficient (K) formula

Answer 3

REFER TO ONENOTE

Question 4

explain analytical chromatography

Answer 4

§ Analysis of complex mixtures
§ Sample volume smaller
§ Shorter columns work
Small scale- molecules are separated + identified but not isolated

Question 5

explain preparative chromatography

Answer 5

§ 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

Question 6

explain the retention factor Rf

Answer 6

REFER TO ONENOTE

Question 7

what are the 4 stages of column chromatography

Answer 7

• Equilibration with buffer
  
  • Sample application elution
  • elution
    Regeneration of column

Question 8

explain ion exchange chromatography

Answer 8

• 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

Question 9

explain cation vs anion exchange chromatography

Answer 9

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

Question 10

explain pI

Answer 10

• 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

Question 11

what factors are proteins seperated by, and what type of techniques can be used for it

Answer 11

• Seperated by
  ○ Charge
  § Ion exchange chromatography

    ○ Size
        § Size exclusion chromatography or gel filtration
  		
    ○ Hydrophobicity
        § Hydrophobic interaction chromatography or reverse phase chromatography
  		
    ○ Affinity Affinity chromatography

Question 12

explain gel filtration/SEC

Answer 12

• 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

Question 13

**what are the important parameters of gel filtration/SEC

Answer 13

○ External volume (void volume) Vo
○ Sum of external + interval volume = total volume, Vt
○ Elution volume, Ve
The partition coefficient in SEC is Kav

Question 14

explain the Kav formula

Answer 14

REFER TO ONENOTE

Question 15

explain the affinity chromatography

Answer 15

• 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

Question 16

explain the affinity chromatography process

Answer 16

1. 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

Question 17

explain the hydrophobic interaction chromatography

Answer 17

• 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

Question 18

explain electrophoresis

Answer 18

• The movement of ions through an electric field
  
  • Proteins seperated by size
  • SDS-PAGE is a type of electrophoresis

Question 19

explain SDS-PAGE

Answer 19

• 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