Anion exchange

chromatography
(AEC)

Anion exchange chromatography

Beads are +vely charged

Eg DEAE-C (Diethylaminoethyl cellulose) matirx
-vely charged proteins bind to beads
+vely charged proteins will elute out
Add buffer to elute unbound proteins
Bound protein are eluted by changing pH or by passing a
gradually increasing linear salt gradient (NaCl high
concentration)
Generally used for acidic proteins
Acidic proteins have low pI
Why for acidic? If you want to use it for basic .very
high pH may denature protein.lose its activity

Matrix or Resin
Matrix Is Support
Covalently attached functional group (charged)
Matrix, polymeric, porous beads
Cellulose
Sepharose: is a tradename for a crosslinked, beadedform of a polysaccharide polymer material extracted
from seaweed. It is crosslinked through lysine side
chains.
Sephacel: bead form of cellulose
Sephadex: crosslinked dextran/bead form of dextran
Packed (poured) into the column as a slurry

IEC Groups
Anion exchange chromatography beads
(matrix/resins)
Carboxymethyl (CM)
(Sulfate derivative)

Quaternary amine

Strong Anion Exchanger

Charged (ionized) across a
wide range of pH levels
Permanantly charged

Diethylaminoethyl (DEAE)

Weak Anion Exchanger

Charged within a
narrower pH range.
Charge dependent on pH
Above 9.0 not good

Commonly Used Anion-Exchange Matrices for Protein Seperation

Cellulose Matrix
Aminoethyl-Cellulose

Exchange Type
Weak

Diethylaminoethyl (DEAE)-Cellulose Weak

Benzyl DEAE-Cellulose

Weak

Polyethylenimine (PEI)-Cellulose

Weak

Diethyl-[2-hydroxypropyl]-aminoehtyl
Strong
(DAE)-Cellulose
Triethylaminoethyl (TEAE)-Cellulose Weak
Diethylaminoethyl (DEAE)-Sephacel Weak

Commonly Used Anion-Exchange Matrices for Protein Seperation

Polystyrene Matrix
Analytical Grade (AG)-1
Analytical Grade (AG)-2
Bio-Rex 5
AG 3-X4A
Bio-Rex MSZ 1-X8

Exchange Type
Strong
Strong
Intermediate
Strong
Strong

Sephadex Matrix
Diethylaminoethyl (DEAE)-Sephadex
Diethyl-[2-hydroxypropyl]-aminoehtyl (DAE)Sephadex

Exchange Type
Weak

Sepharose Matrix
Diethylaminoethyl (DEAE)-Sepharose CL-6B
Diethylaminoethyl (DEAE)-Sepharose (fast flow)
Quaternary amine (Q)-Sepharose (fast flow)

Strong
Exchange Type
Weak
Weak
Strong

Factors considered for choosing IEC matrix

(1) pH Range Where the Protein of Interest is Stable.
If the protein is most stable at pH's above its pI, then an anion-exchange gel matrix should be
used. If it is most stable below its pI, then a cation-exchange gel matrix should be used.
(2) Molecular Size of the Protein being Separated.
The porosity of an ion-exchange matrix affects the binding capacity of a gel matrix. For
proteins of molecular weight 10,000 to 100,000, DEAE-Sephacel and DEAE-Sepharose are
good choices. For proteins less than 30,000, Sephadex A-25 or C-25, which have the highest
charge density at the surface of the gel, are appropriate.
(3) Operating Pressue.
For nonrigid gels (e.g., Sephacel or Sepharose), operating pressures should not exceed the
manufacturer defined limits. For rigid gels (e.g., Analytical grade (AG), Bio-Rex MSZ, and
Dowex commercial grade ion-exchange resins from Bio-Rad), packing can be carried out at
higher flow rates using a peristaltic pump.
(4) Choice of Column Size depends on the Binding Capacity of the Ion-Exchange Gel
Matrix.
The column diameters most frequently used are 1, 2, and 2.5 cm. Reservoir design, direction
of column flow, and whether or not a peristaltic pump should be employed are arbitrary.
Separating columns are usually <20 cm in length. If the protein mixture is exceedingly
complex, a longer column should be used.

Mobile Counter Ions in Anion exchange

Ions that move;
Added in buffer solution
Flow through the column
Interact with charged groups on the beads
Increasing concentration of NaCl added
Cl- is exchanged with protein

Steps of
Anion Exchange chromatography (AEC)
I. Set up column; other equipment
II. Choose buffer (pH important, above protein pI for AEC)
III.Prepare resin; pour column; equilibrate resin with buffer
IV.Load sample onto column --adsorption
V. Wash unbound proteins off the column
VI. UV spectrophotometer= OD 280 nm
VII.Desorption; elution (step gradient/continous gradient)
(Collect fractions of 1ml or 5 ml or 10ml using fraction
collector)
VIII.End desorption; regenerate column (optional)

Analysis of elution profile (chromatogram)

Analyze sample for purity on SDS-PAGE

Pool fractions containing pure samples

Chromatogram

What MakesProteins Adsorb

And Desorb From The Column?
Proteins have charges because
their amino acids are charged
At a given pH, different proteins
have different charges, because
they are composed of different
amino acids

Charge On Proteins
Overall net charge on a protein is
determined by the R groups of its
amino acids
Some amino acids have R group with
positive charge, others negative
charge, others no charge

In theory, IEC can separate

proteins that differ by only one
charged group

Or
Two protein both negatively
charged will bind anion exchange
matrix !!!!!!!!!!
Can both be separated?

pH Of Environment
Is Crucial

pH affects stability of proteins

pH of environment affects charge on
a protein
pH is controlled with the buffer
chosen

pI Is Important
First, pI for a protein of interest
determines whether use cation or
an anion resin

pI
Also, usually dont want to use a pH
where the protein has no charge at
all because it will never stick to
the beads
So, for example, pI is 6.2, may want
to use a buffer at 7.2.

IEC Principles

Order of elution: +vely will not bind and negatively charged protein will bind

IEC Principles
Mehods of Elution / desorption of
proteins bound to anion exchange
With Salt Gradient:
Counter anion Chloride ions (NaCl)
Increasing concentration
First least negatively charged proteins will elute
Followed by more negaitvely charged proteins
OR
With pH gradient
Lower the pH slowly
Proteins will slowly become less negative and
than positive as pH will reach below its pI

Practice

Next lecture

Cation exchange
chromatpgraphy (CEC)