L12+13 - Chromatography and Electrophoresis Flashcards
Mobile and stationary phase:
Chromatography:
Mobile phase is the mixture of substances that needs to be separated.
Stationary phase is the solid porous matrix.
Chromatography separates proteins.
Physical properties:
They all have different physical properties so some bind more strongly than others.
Have different interactions with stationary phase so separates proteins. Weak ones are eluted first.
Separating methods:
Solubility, ionic charge, polarity, molecular size, binding specificity:
Solubility:
Salting in and salting out.
Ionic charge:
Ion exchange chromatography
Electrophoresis
Isoelectric focusing
Polarity: Adsorption chromatography Paper chromatography Reverse-phase chromatography Hydrophobic interaction chromatography
Molecular size: Dialysis and ultrafiltration Gel electrophoresis Gel filtration chromatography Ultracentrifugation
Binding specificity:
Affinity chromatography
Ion exchange-chromatography:
Ions that are electrostatically bound to insoluble and chemically inert matrix can be reversibly replaced by ions.
R+A- + B- = R+B- + A-
Anion exchanger
R-A+ + B+ = R-B+ + A+
Cation exchanger
Separates according to charge. If resin is positive the negative proteins bind and the positive ones are eluted first. More -ve the charge then the stronger the interaction.
Interaction depends on conc of ion.
More ion = weaker interactions.
Separate protein from resin by increasing salt conc.
Also interactions depends on pH.
pH pI protein has negative charge.
Different pI of protein so different charges at certain pH.
Stepwise elution:
Separate by increasing salt conc. Weaker electrostatic interactions when you increase it. Proteins eluted at different times.
Gradient elution:
Set up gradient of salt conc by mixing two buffers of different salt conc. Low salt -> high salt.
Gel filtration chromatography:
Separates according to size and charge.
Resin beads have pores and small molecules can enter and so are eluted slower than large ones which can’t enter.
Pore size varies.
Resin bead has gel matrix which slow down speed of molecules.
Gel’s exclusion limit:
Smallest molecule that is unable to penetrate pores of a given gel.
Elution volume of a given solute (Ve):
Volume of solvent required to elute solute from column.
Void volume (Vo):
Volume of solvent space surrounding beads and can be easily measure.
It is to know if you have to use large/small column.
Relative elution volume (Ve/Vo):
Can use this to estimate molecular mass of unknown proteins by plotting against known ones (kD).
Large Ve/Vo means smaller molecule so is eluted first.
Affinity chromatography:
Separates according to their ability to bind to specific molecule. Depend on specific interaction between two proteins: antigen and antibody.
Resin has specific antibody so only specific antigen binds covalently. Others are eluted.
Increases yield as it is more purer. Get specific product.
Reverse-phase chromatography:
Separates by polarity - separates polar ones. Proteins denature when exposed to hydrophobic environments. Unfold and expose hydrophobic side chains.
Liquid-liquid chromatography.
Stationary phase - liquid immobilised with C8 and C18 alkyl chains.
Mobile phase - polar liquid.
To break interaction of protein and resin, elution phase (solvent to remove proteins) should be highly non polar.
Hydrophobic interaction chromatography (HIC):
Doesn’t allow protein denature like RPC.
Stationary phase - hydrophilic substance with hydrophobic groups. Forms weak interactions and maintains protein fold.
Can elute by weakening hydrophobic interactions e.g. Decrease salt conc. (Ion exchange is opposite).
Electrophoresis:
Analyse fractions eluted from chromatography.
Study macromolecules and separate them.
It is the migration of ions in electric field.
