CHEMISTRY PRESENTATION:ION EXCHANGE CHROMATOGRAPHY BY: LEOR SURILOV

TABLE OF CONTENT

Protein Overview: Why do proteins have a net charge? What is Ion Exchange Chromatography? The two types of Ion Exchange Chromatography So how do the proteins move down the column? Overview of the steps Steps of Ion Exchange Chromatography Factors affecting Ion Exchange Chromatography Why is Ion Exchange Chromatography important? How is it related to chemistry? Conclusion References

PROTEIN OVERVIEW: WHY DO PROTEIN HAVE A NET CHARGE?

Proteins are composed of 20 types of amino acids These amino acids differ from each other based on their side chains These side chains can be: positively charge, negatively charged, and uncharged:

Positively Charged Side Chain: Lysine, Arginine, and Histidine Negatively Charged Side Chain: Aspartate and Glutamate

Proteins are a long strand of varying amino acids creating a net charge value

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WHAT IS ION-EXCHANGE CHROMATOGRAPHY?

Chromatography is a method of purification There are various constituents within the solution and the ability to purify the

desired constituent is important Ion-exchange chromatography can be used to purify mixture of proteins

based on their net charge The Ion-exchange separation is done by having a resin that is charged

opposite to the desired constituent/protein

http://chemwiki.ucdavis.edu/@api/deki/files/223/=ion_exch.jpg?revision=1

THE TWO TYPES OF ION-EXCHANGE CHROMATOGRAPHY Two types of setups that begin with a funnel placed on top of the column Inside a narrow column is special gel beads/surface These beads can be two types called:

Anion Exchange Chromatography: Immobilized cation surface/beads (commonly used anion exchange resins are Q-resins, amines, and DEAE resins)

Cationic Exchange Chromatography: Immobilized anion surface/beads (commonly used cation exchange resins are S-resins, sulfate derivatives, carboxylate derived ions)

The type of beads that are chosen are based on if your protein is net positive or negative

The bead are mostly made of some sugar polymer ie. cellulose

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SO HOW DO THE PROTEINS MOVE DOWN THE COLUMN

Gravity separates the proteins in the column based on the charge Proteins with the greatest net positive or negative (depending on the beads used) will move the slowest down the

column because they will be attracted to the beads strongly The other proteins either slightly positive/negative will be less tightly bound The proteins that are the same charge as the resin will travel down the column the quickest Therefore we can separate proteins based on their varying charge= purification

OVERVIEW OF THE STEPS

The protein mixture is poured into the funnel The protein mixture will then begin at the top of the column The gravitational forces cause them to move down the column. The differences in net charge causes the proteins to move down the column at different

rates The protein with the same charge as the beads will travel down the fastest, while the

proteins opposite of the beads will separate based on the strength of the positive charge Pouring a salt solution in the end we can break the electric interactions between the

beads and the desired proteins, leading it to finally elute

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STEPS OF ION-EXCHANGE CHROMATOGRAPHY

The basic process of chromatography follows these steps:

1. Equilibrium: the equilibration of the stationary phase to the desired start conditions. When equilibrium is reached all stationary phase charged groups are bound to a counter-ions (ie. NaCl)

2. Sample Application: The goal here is to bind the target molecule/s and wash out all unbound material. The sample buffer should have the same pH and ionic strength as the starting buffer in order to bind the desired charge proteins

3. Elution: Is the biomolecules being released from the ion exchanger by a change in the buffer composition. A common way of achieving this is to increase ionic strength with sodium chloride, in order to separate the bound proteins. Proteins are separated based on the number of charged groups on their surface

4. Regeneration: The final step is to remove all molecules still bound. This is done to ensure that the column is able to run the stationary phase properly when used again using final high strength wash

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FACTORS AFFECTING ION EXCHANGE CHROMATOGRAPHY

Resin Properties The cross-linking should allow charge molecules to be affected not the size of the molecule.

pH of Mobile phase (Buffer) – main disadvantage Nature of the Exchanger ions (valence of ions can play a big role in separations) Proteins charge similarity (the net charge of the desired protein has to have a big difference and won’t work if

they have the same or similar net charge then ion exchange chromatography will not work or if there is a net neutral charge)

WHY IS ION EXCHANGE CHROMATOGRAPHY IMPORTANT?

It is one of the most common types of chromatography Can be applied for separation and purification of many charged or ionizable molecules such as proteins, enzymes,

nucleotides, DNA, antibiotics, vitamins etc. because many molecules are generally charged Clinical Application: Enzymes, Isoenzymes (N-acetyl-ß-D-glucosaminidases have been used to diagnosis

haematological malignancies), Immunoglobulins etc. Industrial Application: Water and wastewater treatment Drug Development: Used to improve stabilities and solubility properties of active drugs

HOW IS THIS RELATED TO CHEMISTRY?

Electrostatic attraction for anions and cations are based on the strength of the positively and negatively charged groups

You must understand pH, buffers, and salt interaction because the have a big impact on protein binding

At a certain pH called the isoelectric point, the positive and negative charges on the amino acid are equal and the overall charge is zero http://www.periodni.com/gallery/isoelectric_point.png

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CONCLUSION

Ion Exchange Chromatography is a purification technique that separates proteins based on charge There are two types of Ion Exchangers: Anion and Cation The process involves the binding of desired constituent to the oppositely charged resin It is related to chemistry because it involves chemical interactions and buffer systems namely the isoelectric point,

which involves pKa values It is used in a variety of industries and fields because of the simplicity of purification technique

REFERENCES

Cation Exchange Chromatography. (n.d.). Retrieved July 18, 2016, from http://www.bio-rad.com/en-ca/applications-technologies/cation-exchange-chromatography

Ion Exchange Chromatography. (n.d.). Retrieved July 18, 2016, from http://www.sigmaaldrich.com/life-science/proteomics/protein-chromatography/ion-exchange-chromatography.html

Kaplan MCAT Organic Chemistry Review. (2016). Larson, D. L. (2017). Clinical chemistry: Fundamentals and laboratory techniques. St. Louis:

Elsevier. Principles of Ion Exchange Chromatography. (n.d.). Retrieved July 18, 2016, from

http://www.separations.us.tosohbioscience.com/ServiceSupport/TechSupport/ResourceCenter/PrinciplesofChromatography/IonExchange