Lecture 2 - Ion Exchange Chromatography

what are the six steps to purifying a protein?

1. Extraction of crude protein preparation from source material 2. concentration and initial purification 3. chromatographic purification (if req) 4. stabilization of final product and adjustment of activity to required level 5. drying of product (if req) 6. final product packaging and labelling

name the five main stages of an ion exchange experiment

1. equilibration 2. sample application and adsorption 3-4. substances are removed from the column by changing elution conditions 5. regenerate resin by adding a counter-ion

what are the three principal types of adsorption chromatography for enzyme purification?

1. ion-exchange chromatography 2. affinity 3. gel exclusion/gel filtration chromatography/molecular sieve

what are the two types of chromatographic methods?

1. partition chromatography 2. adsorption chromatography

absorbance at ... nm can be to used to monitos amino acids/proteins (tryptophan and tyrosine)

280

give the formula for relative mobility

Rf = 1 - alpha

cation exchange matrices have anionic functional groups such as...

SO3-, OPO3-, COO- (most popular is CM)

what kind of column is Dowex-1?

an anion exchange (quaternary amine) resin

describe affinity chromatography

an antibody or ligand / specific binding based

..... is the total volume of solvent and adsorbing material taken up by the column

bed volume

name two weak cation exchange functional groups

carboxyl (COO-) and carboxymethyl (CH2COO-), used in cellulose, sephadex and agarose

anion exchange matrices usually contain...

cationic tertiary and quaternary ammonium groups (most popular is DEAE)

the first ion exchangers designed for use with biological substances were .... since they would not denature proteins due to ....

cellulose ion exchangers, hydrophilic nature (but they had low capacities and were unstable)

cellulose, polystyrene, agarose and cross-linked dextran resins are usually used in purification for...

charge separation

name a weak anion exchange funcitonal group

diethylaminoethyl DEAE (CH2CH2NH(CH2CH3)2), used in cellulose, sephadex and agarose

chromatography separates molecules based on...

differential migration through a porous medium

once a mixtrue of compounds is loaded at the top of the solid matrix, they can be...

differentially eluted or developed

describe partition chromatography

distribution of a solute between two liquid phases (can involve extraction using two liquids or a liquid immobilized on a solid support)

how can you calculate Kb (partition coefficient)?

divide concentration of the solute in the stationary phase (Cs) by the concentration of solute in the mobile phase (Cm)

movement of solvent through loaded column is called ....

eluting

describe the third and fourth stages of an ionic exchange experiment: substances are removed from the column

elution conditions are changed: usually increasing of the ionic strength of the buffer or changing the pH

gel exclusion chromatography is usually the .... step of purification

final

as material emerges or elutes from the column, the eluate can be separated into....

fractions

what are the two types of elution using a salt concentration gradient?

gradient elution or step elution

the greater the values of the partition coefficient, the .... the affinity of the compound for the stationary phase

greater

why is it important to apply a small amount of sample to the top of the resin bed?

if too large, the compounds will enter in a diffuse manner and we will get zone spreading

cross-linked agarose (sepharose CL-6B) and cross-linked cellulose (DEAE Sephacel) were the first ion exchange matrices to combine a spherical form of beads with high porosity, leading to...

improved flow properties and high capacities for macromolecules

in partition chromatography, the stationary phase consists of...

inert solid particles coated with liquid adsorbent (relies on nonspecific solubility factors)

ion-exchange and affinity chromatography can handle large quantities of crude enzyme, but _____ are prefered for earlier stages of purification

ion-exchange materials (since they are cheaper)

adding sample to a column is called ...

loading

describe adsorption chromatography

porous matrix or solid support is enclosed in a cylinder/column saturated with aqueous buffer or organic solvent

in solutions of pH below their isoelectric point, proteins will be .... charged and bind to ..... exchangers

positively, cation

when the adsorbing material is made into a column it is said to be .... or .....

poured or packed

name a strong anion exchange functional group

quaternary amine QAE or other amines (NH2+), used in sephadex or dextran

what does ninhydrin do?

reacts with amino acids to produce a purple/pink colour

describe the second stage of an ionic exchange experiment: sample application and adsorption

sample carrying the appropriate charge displace counterions binds reverisbly to the gel, while unbound molecules are washed out

describe gel exclusion / gel filtration chromatography (or molecular sieve)

separates proteins or amino acids based on size, since smaller proteins travel through the column more slowly than larger proteins

what is chromatography used for?

separating molecules based on chemical properties like molecular mass, charge, solubility or affinity for a particular ligand or solid support

sephadex (cross-linked dextran), sepharose (cross-linked agarose), and bio-gel (cross-linked agarose) have a high loading capacity and are used for...

separation based on charge and size

adsoption chromatography relies on...

specific interactions between the solute molecules and binding sites on the stationary phase (can be ionic, h-bonding, hydrophobic or ligand interaction, but are always reversible)

adsorption chromatography uses a .... phase

stationary phase (finite number of specific binding sites for the solute molecules)

what is the difference between strong and weak ion exchange resins?

strong maintain a net charge at extremes of pH (but form stable salts with strongly ionized counter ions), weak are subject to titration at extremes, since the charge on the beads can change

name two strong cation exchanger functional groups

sulpho (SO3-) and sulfopropyl (CH2CH3CH2SO3-), used in sephadex, cellulose and dextran

what is a partition coefficient (Kb)?

the affinity of a compound for the stationary phase, ie the fraction of the compound that is adsorbed on the stationary phase at any given point in time (value between 0-1)

what is relative mobility?

the affinity of a molecule for the mobile phase, describes the rate of migration of the molecules relative to the rate of migration of the mobile phase passing through the solid support

define the elution volume

the amount of solvent required to remove a particular solute (sample) from a column (remove what is loaded, equivalent to Rf values in TLC)

describe the first stage of an ionic exchange experiment: equilibration

the ion exchanger is brought to a starting state, in terms of pH and ionic strength

what is alpha in terms of a partition coefficient?

the molecules adsorbed on stationary phase divided by the molecules in stationary and mobile phase

desribe the fifth and final stage of an ionic exchange experiment

the resin is regenerated by adding a counter-ion

in the introduction of an increasing salt concentration gradient, solute molecules are released form the column in the order of....

their strengths of binding

one disadvantage of resins and gel-type exchangers is that....

they tend to become compressed at high pressure, greatly reducing flow rate

what is the principle behind separation through chromatography?

a mixture of compounds will have different affinities for the stationary phase on which it is adsorbed and the mobile phase passing through the stationary phase

preparative and analytical chromatography is usually performed in...

a column format (adsorption chromatography)

describe ion-exchange chromatography

a column is packed with beads that have a positive or negative charge, so proteins with the opposite charge bind to the beads (can be eluted by passing a salt gradient, which desorb/outcompete the protein)

why must ionic strength be taken into account when doing ion exchange chromatography with a protein?

too low and the protein will not be soluble, too high and the salt can compete with the protein for ion-exchange sites

.... is the volume taken up by the liquid phase

void volume

ion-exchange materials are generally....

water insoluble polymers containing cationic or anionic groups