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Ion-Exchange Chromatography roduction to Chromatography: General Princip -Exchange Chromatography Principles -Exchange Chromatography Resins Charge of Amino Acids, Peptides, and Protei egrated and Automated Systems Reading: N & B Ch. 5

Ion Exchange Chromatography Lecture

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Page 1: Ion Exchange Chromatography Lecture

Ion-Exchange Chromatography

Introduction to Chromatography: General Principles

Ion-Exchange Chromatography Principles

Ion-Exchange Chromatography Resins

Net Charge of Amino Acids, Peptides, and Proteins

Integrated and Automated Systems

Reading: N & B Ch. 5

Page 2: Ion Exchange Chromatography Lecture

Chromatography literally means “color writing”.

Chromatography was invented by the Russianbotanist Mikhail Tsvet in 1900. He used it to separate chlorophyll-containing extracts of plants.

Key idea is that molecules of interest interactdifferentially with the stationary phase and a mobilephase, and thus can be separated.

Introduction

Page 3: Ion Exchange Chromatography Lecture

The Basic Principle

Page 4: Ion Exchange Chromatography Lecture

Partition Coefficient and Relative Mobility

Partition coefficient describes the affinity of a compound for the stationary phase.

α or (Kav)= molecules adsorbed on stationary phase molecules in stationary and mobile phase

Can have values between 0 and 1. Example, a molecule with α = 0.4 will be 40% adsorbed on the stationary phase.

Relative mobility or retention factor (Rf) describes the affinity of a molecule for the mobile phase.Rf = 1 – α (Recall Rf from TLC in Organic Chem)

Page 5: Ion Exchange Chromatography Lecture

Ion-Exchange ChromatographySeparates molecules on the basis of charge

Beads of the resin modified so that they containa cationic or anionic functional group that can bepositively charged, negatively charged, or neutraldepending on pH.

A solution that contains the species of interest is applied to the column containing the resin, and thesample either binds to the resin or passes throughthe column. A gradient (e.g., salt or pH) can then beused to elute the desired compound if the compoundadhered to the resin.

Page 6: Ion Exchange Chromatography Lecture

Ion-ExchangeChromatography

Would the resin inthis example beconsidered ananion-exchangeresin or a cation-exchange resin?

Page 7: Ion Exchange Chromatography Lecture

Ion Exchange Resins

Proteins with net negative charges (excess of negative charges) adsorb to anion exchangers, while those with net positive charges (excess of positive charges) adsorb to cation exchangers. The strength of the adsorption increases with increased net charge.

Page 8: Ion Exchange Chromatography Lecture

Ion-ExchangeChromatography:a closer look

Page 9: Ion Exchange Chromatography Lecture

http://www.expasy.ch/spdbv/images/1YDRsurf.jpg

Electrostatic Potential Map of the Surface of a Protein

Page 10: Ion Exchange Chromatography Lecture

DesorptionTwo possibilities exist to desorb sample molecules from the ion exchanger:

1. Reducing the net charge by changing pH. 2. Adding a competing ion to "block" the charges on the ion exchanger.

Page 11: Ion Exchange Chromatography Lecture

Principle of Ion-Exchange Chromatography

Fig. 5-13 (Ninfa & Ballou)

Page 12: Ion Exchange Chromatography Lecture

Principle of Ion-Exchange Chromatography cont.

Fig. 5-13 (Ninfa & Ballou)

Page 13: Ion Exchange Chromatography Lecture

What is the nature of the functional groups that arecovalently linked to the resin?

You will be using DE52, whichcontains DEAE functional groupsattached to a cellulose matrix

Page 14: Ion Exchange Chromatography Lecture

Depending on the pKa value of the charged ligand, the ion exchangers are divided into strong and weak.

Strong ion exchangers are fully charged over the total pH range normally applicable to proteins and peptides.

With weak ion exchangers, the charge displayed is a function of the eluent pH.

Page 15: Ion Exchange Chromatography Lecture

Examples

Strong anion exchangers-CH2N+(CH3)3 trimethylaminoethyl TAM-C2H4N+(C2H5)3 triethylaminoethyl TEAE

Weak anion exchangers-C2H4N+H3 aminoethyl AE-C2H4N+(C2H5)2 diethylaminoethyl DEAE

Strong cation exchangers-SO3

− sulpho S-CH2SO3

− sulphomethyl SM

Weak cation exchangers-CH2COO− carboxymethyl CMadapted from N & B Table 5-2

Page 16: Ion Exchange Chromatography Lecture

How Do We Know If “Our” Protein Is Goingto Bind the Ion-Exchange Resin That We AreUsing? – pH, pKa, pI & Buffers Revisited:

pH = −log[H+] (not strictly true but a useful, workingdefinition)

pH = pKa + log([basic form]/[acidic form]) [HH eq]

Isoelectric point (pI) is the pH at which a molecule has a net charge of zero.

Buffers useful ±1 (or ±0.5) units above and belowtheir pKa

Page 17: Ion Exchange Chromatography Lecture

We start by considering a simple, weak acid: RCOOH

Then, we consider a weak base: RNH2

Then, we will consider a compound that has both of the above functional groups – i.e., an amino acid

Then, we will consider small peptides

Finally, we will extrapolate to a polypeptide – i.e., aprotein

This discussion will require some board work

Deciding on the Charge of Our Protein:

Page 18: Ion Exchange Chromatography Lecture
Page 19: Ion Exchange Chromatography Lecture

Group pK

a

N-terminal amino

8.0

C-terminal carboxyl

3.1

Asp, Glu 4.0

Lys 10.4

Arg 12.5

His 6.0

Tyr 10.5

Cys 8.4

The pKas of groups or side chains can and do varysomewhat from what their values are in free aminoacids. The values in the table below are meant to be approximate, but on average, fairly representative

Page 20: Ion Exchange Chromatography Lecture

http://ca.expasy.org/tools/pi_tool.html

http://emboss.sourceforge.net/

(and a number of other sites)

Determination of pI for a Protein

Or, experimentally determine pI by using isoelectric focusing, a topic we will take up when we discuss SDS PAGE.

Page 21: Ion Exchange Chromatography Lecture

Pharmacia handbook

Now that we understand the concept of pI (I hope),we are in a better position to consider the choice of ion exchanger

Page 22: Ion Exchange Chromatography Lecture

The pH vs. net surface charge curves for three different proteins are shown. Schematic chromatograms for a CM and a DEAE ion exchanger are shown at the top and bottom, respectively.

Page 23: Ion Exchange Chromatography Lecture

Proteins are usually least soluble and often precipitate at their isoelectric point.

WHY?

A Question to Ponder

Page 24: Ion Exchange Chromatography Lecture

Conditions used to purify a protein are oftendetermined empirically. You likely will choose which resin to use on the basis of the pI of the protein (if it is known or can be estimated). Then you need to decide on the buffer, the salt, the steepness of the gradient, etc… You may want to run some pilot experiments.

After you decide which resin you want to use,you will then have to:

-swell (hydrate) of the resin -load the sample-equilibrate with buffer -elute the sample-pack or pour the column -locate the sample-equilibrate the sample -determine purity

Considerations

Page 25: Ion Exchange Chromatography Lecture

Gradients of a neutral salt are formed by mixing two eluents, one containing a low concentration of the neutral salt (buffer A) and one containing a high concentration of this salt (buffer B). But for their salt contents, the two eluents are identical. Chromatography systems usually control the gradient formation by the use of two pumps, one for buffer A and one for buffer BAmersham Biosciences

Gradient Elution

Page 26: Ion Exchange Chromatography Lecture

low salt

high salt

Fig. 5-14 (Ninfa & Ballou)

A simple gradient maker:

In our ion-exchangechromatography lab, we will not use a gradient. Rather, we willuse a step elution in which we go from low salt to highsalt in one step

Page 27: Ion Exchange Chromatography Lecture
Page 28: Ion Exchange Chromatography Lecture

Steep vs. shallow gradient elution

Page 29: Ion Exchange Chromatography Lecture

Amersham Biosciences

Steep vs. Shallow Gradient Elution – Another View

The distance between peaks is controlled by the slope of the gradient

Page 30: Ion Exchange Chromatography Lecture

HPLC(High Performance or High-Pressure Liquid Chromatography)

FPLC(Fast Protein or Fine Performance Liquid Chromatography)

A Bit More On Integrated and Automated Chromatography Systems

Page 31: Ion Exchange Chromatography Lecture

Schematicof an automatedsystem (FPLC)

Sample load

Pumps

Fraction collector

Low salt High salt

Sheehan, David (2003). Fast Protein Liquid Chromatography. 244. pp. 253.