chromatography

mobile phase

liquid or gaseous fluid percolated through the stationary phase which the mixture is dissolved into

stationary phase

a column consisting of a porous solid matrix

column chromatography

solvent continuously applied to the top of column from a large reservoir of solvent, sample loaded onto the top of the column, as the mobile phase moves down, compounds with a weaker affinity for the stationary phase move down faster and exit the column first where they are collected
stationary phase: solid pourous absorbant materia, usually silica gel or alumina
mobile phase: liquid solvent

how would you separate a mixture by its components’ solubility?

1. salting in

2. salting out

how would you separate a mixture based on its components’ ionic charge?

1. ion exchange chromatography

2. electrophoresis

3. isoelectric focusing

how would you separate a mixture based on its components’ polarity

1. adsorption chromatography

2. paper chromatography

3. reverse-phase chromatography

4. hydrophobic interaction chromatography

how would you separate a mixture based on its components’ molecular size?

1. dialysis and ultrafiltration

2. gel elctrophoresis

3. gel filtration chromatography

4. ultracentrifugation

how would you separate a mixture based on its components’ binding specificity?

affinity chromatography

ion exchange

ions that are electrostatically bound to an insoluble and chemically inert matrix are reversibly replaced by ions in solution

R⁺A^- + B^- ⇌ R⁺B^- + A^-

R+A- is an anion exchanger

R^-A⁺ + B⁺ ⇌ R^-B⁺ + A⁺

R-A+ is a cation exchanger

ion exchange chromatography

liquid mobile phases is passed through a charged stationary phase, different proteins have different affinity for the ion exchanger depending on their net charges

what factors dictate the affinity of a protein (polyelectrolyte) to an ion exchanger and why

identity and concentration of other ions in solution causing competition

pH because the net charges of acid-base groups vary with pH - when pH<pI +ve, when pH>pI -ve

stepwise elution

gradient elution

gel filtration/size exclusion chromatography

molecules are separated according to their size and shape, smaller molecules enter the pores of the gel beads of the column and large molcules are eluted in a smaller eluant volume

gel filtration/size exclusion chromatography

size exclusion chromatography

gel exclusion limit

the smallest molecule unable to penetrate the pores of a given gel

elution volume of a given solute (V_e)

the volume of solvent required to elute the solute from the column

void volume (V₀)

the volume of solvent space surrounding the beads - can be easily measured

relative elution volume, what can it be used to estimate?

elution volume/void volume

used to estimate molecular masses of each solute

affinity chromatography

a ligand that binds specifically to a protein of interest is attached covalently to a resin, an impure protein solution is passed through and the desired protein non-covalently binds to the ligand and the others are washed away

the desired protein is then released changing elution conditions

affinity chromatography

immunoaffinity chromatography

type of affinity chromatography, uses antibodies

glutathione-S-transferase

type of affinity chromatography, uses glutathione

insulin affinity chromatography ligand

insulin receptor

glucose affinity chromatography ligand

glucose binding protein

metal chelation affinity chromatography ligand

His-tag and divalent metal ions (Ni2+, Co2+, Zn2+)

reverse phase chromatography

liquid-liquid, used to separate non-polar substances
stationary phase: liquid immobilised on silica beads substituted with C8 and C18 alkyl chains (non-polar)
mobile phase: MORE polar liquid
eluting mixture: less polar liquid

retention time

time at which a compound emerges from the column

factors which effect retention time

hydrophobicity, number of carbon atoms, branched or unbranched, saturated or unsaturated, functional groups

what affect on retention time does hydrophobicity have?

more hydrophobic longer time

what affect on retention time does the number of carbon atoms have?

longer C chain longer time

what affect on retention time does branched/unbranched have?

unbranched takes longer

what affect on retention time does saturated/unsaturated have?

saturated takes longer

what affect on retention time do functional groups/charges have?

neutral polar and charged species elute earlier, then acidic compounds

how can we increase retention time?

add salts to the mobile phase
add more water to mobile phase
lower pH of mobile phase

hydrophobic interaction chromatography (HIC)

separates native proteins by surface hydrophobicity
stationary phase: hydrophilic lightly substituted with hydrophobic groups (octyl or phenyl residues)

how does hydrophobic interaction chromatography work?

hydrophobic amino acid side chains on the surface of proteins will interact with the column, resulting in weak interactions that maintain the native fold of the protein

how is elution achieved in hydrophobic interaction chromatography

progressivey weakening the hydrophobic reactions, e,g. aqueous solutions with decreasing salt concentrations

how is hydrophobic interaction chromatography different to reverse phase chromatography?

ligands in reverse phase chromatography are much more hydrophobic than HIC ligands

therefore we can use more moderate elution conditions in HIC that do not disrupt the sample as much

high performance pressure liquid chromatography

use pumps to apply sample under high pressure, not reliant on gravity

columns made of smaller adsorbent particles (2-50um) giving superior resolving power

pros of HPLC

can use smaller sample amounts

not reliant on gravity and automated

small adsorbent particles give superior resoling power

high speed and sensitivity