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=Protein Separations - Chromatography- Table of Contents=
#[[Proteomics/Protein Separations - Chromatography| Protein Separations - Chromatography]]
#[[Proteomics/Protein Separations - Chromatography/Chromatography Theory| Chromatography Theory]]
#[[Proteomics/Protein Separations - Chromatography/Stationary and Mobile Phases| Stationary and Mobile Phases ]]
#[[Proteomics/Protein Separations - Chromatography/Ion exchange|Ion exchange]]
#[[Proteomics/Protein Separations - Chromatography/Molecular Exclusion|Molecular Exclusion]]
#[[Proteomics/Protein Separations - Chromatography/Affinity|Affinity]]
#[[Proteomics/Protein Separations - Chromatography/Immobilized Metal Ion Affinity Chromatography (IMAC)|Immobilized Metal Ion Affinity Chromatography (IMAC)]]
#[[Proteomics/Protein Separations - Chromatography/Reversed Phase|Reversed Phase]]
#[[Proteomics/Protein Separations - Chromatography/Hydrophobic Interaction Chromatography (HIC)|Hydrophobic Interaction Chromatography (HIC)]]
#[[Proteomics/Protein Separations - Chromatography/Two Dimensional (2D) Chromatography|Two Dimensional (2D) Chromatography]]
#[[Proteomics/Protein Separations - Chromatography/Ultra High Performance Liquid Chromatography|Ultra High Performance Liquid Chromatography]]
-->
Chapter written by: Laura Grell and Alexander Butarbutar <br>
Contact llg3875@rit.edu or nbb3924@rit.edu for contributions <br>
Chapter modified by Kai Burnett and Dalia Ghoneim <br>
Contact kab9783@rit.edu or dxg6098@rit.edu
==Ion Exchange Chromatography==
[[Image:anion+cation exchanger2.GIF|thumb|right|300px|Figure 1. An anion and cation exchanger]]
&nbsp;&nbsp;&nbsp;Ion exchange is probably the most frequently used chromatographic technique for the separation and purification of proteins, polypeptides, nucleic acids, polynucleotides and other charged biomolecules (Ref1). The reasons for the success of ion exchange are its widespread applicability, its high resolving power, its high capacity and the simplicity and controllability of the method [http://sbio.uct.ac.za/Sbio/documentation/Ion_exchange_chromatography.pdf (Ref2)]


&nbsp;&nbsp;&nbsp;Just as with other forms of chromatography, ion exchange chromatography utilizes both a stationary and mobile phase. The stationary phase in this method carries either a positive or negative charge. The charged stationary phases are named according to the types of charged particles that bind to them. 


The stationary phase for anion exchange consists of a synthetic polymer (resin) which contains many positively charged functional groups, typically quaternary ammonium ions. Negatively charged species are attracted to the resin and travel more slowly down the column.  Similarly, positively charged species are attracted to a cation exchange resin's negatively charged functional groups. Sulfonate groups are commonly found as side-chains in cation-exchange resins.  The stationary phases are available as granular material or beads, but prepacked column are now readily available : [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&ParentId=702507&moduleid=166664 GE HealthCare], [http://www.sigmaaldrich.com/Brands/Aldrich/Tech_Bulletins/AL_142/Sephadex_Products.html Sigma-Aldrich], [http://www.rohmhaas.com/ionexchange/  Rohm and Haas], [http://www.ion-exchange.com/ Sybron Chemicals], [http://www.edvotek.com/pdfs/243.pdf Edvotek Ion Exchange Chromatography Kit], [http://www.bio-rad.com/B2B/BioRad/product/br_category.jsp?BV_SessionID=@@@@0521625230.1147406709@@@@&BV_EngineID=ccccaddhkghhhihcfngcfkmdhkkdfll.0&divName=Process+Separations&loggedIn=null&serviceLevel=null&lang=English&csel=null&catLevel=4&catOID=-13554&isPA=false&categoryPath=Catalogs%2fProcess+Separations%2fIon+Exchange%2fIon+Exchange+Chromatography Bio-Rad]. The particles that bind to the stationary phase can be released by changing the buffer conditions.

==Charge On Protein==
&nbsp;&nbsp;&nbsp;The charge on the protein affects its behavior in ion exchange chromatography. Proteins contain many ionizable groups on the side chains of their amino acids including their amino - and carboxyl - termini. These include basic groups on the side chains of lysine, arginine and histidine and acidic groups on the side chains or glutamate, aspartate, cysteine and tyrosine. The pH of the solution, the pK of the side chain and the side chain’s environment influence the charge on each side chain. The relationship between pH, pK and charge for individual amino acids can be described by the Henderson-Hasselbalch equation, which is described in detail elsewhere:

&nbsp;&nbsp;&nbsp;In general terms, as the pH of a solution increases, deprotonation of the acidic and basic groups on proteins occur, so that carboxyl groups are converted to carboxylate anions (R-COOH to R-COO-) and ammonium groups are converted to amino groups (R-NH3+ to R-NH2). In proteins the isoelectric point (pI) is defined as the pH at which a protein has no net charge. When the pH > pI, a protein has a net negative charge and when the pH < pI, a protein has a net positive charge. The pI varies from protein to protein.  This is the reason ion exchange is useful for separating proteins.  If a buffer containing more than one protein is used with an anion exchange resin, then the most negatively-charged protein will be most attracted to the stationary phase and will therefore elute last and the protein with the highest positive charge will elute first.

==Stages in Ion Exchange Chromatography==

The Ion Exchange process can be separated into four basic stages (Res1):

# Equilibration
# Application of sample
# Elution
# Regeneration

[[Image:iex.png]]

===Equilibration===
This stage involves setting up the desired starting conditions so that the system is ready for the ion exchange process. In this step the a buffer with the desired conditions is applied, and all of the charged group in the stationary phase are associated with ions of the opposite charge. When this process in complete, equilibrium has been reached.

===Application of Sample===
In this step, the sample is applied to the stationary phase. Only proteins carrying a charge opposite to the stationary phase will bind to it while those with the same charge or no charge will not bind. These unbound particles will wash out during this stage. In the chromatogram above, protein A never binds to the stationary phase and washes out forming the first peak.

===Elution===
This step involves changing the buffer conditions to particles that have bound to the stationary phase. There are several different ways to do this. One option is to change the pH of the buffer solution. As mentioned before, when the pI of a protein  is the same as the pH of a solution, the net charge of the protein will be zero. Therefore, when the buffer solution's pH reaches the pI of the protein, the protein's net charge will be zero. The protein will no longer bind to the stationary phase and will be released and washed out. Another method to elute the bound protein is to increase the salt concentration. As the salt concentration of the buffer increases, salt ions replace the bound protein. Proteins with weaker ion interactions will be released at lower salt concentrations. Proteins with stronger charges will have a higher affinity for the stationary phase and will remain bound to the column longer. In the chromatogram depicted above, protein B is released due a change in the conditions. The red line indicates some gradient applied to the system. When protein B is released, a second peak is created.

===Regeneration===
This step simply involves removing all bound protein from the stationary phase so that it is ready for another process.

==Anion and Cation Exchangers==

===Anion Exchangers===
[[Image:Anions.png|thumb|right|600px|Figure 2. Anion Exchanger - [http://www.proteinchemist.com Proteinchemist.com]]]
&nbsp;&nbsp;&nbsp;While the resin involved with an anion exchanger is positively charged, anion exchangers are named so because of their affinity to anions.  To effectively bind proteins, the pH of the buffer in the system must be greater than the isoelectric point of the protein of interest, as proteins are negatively charged above their isoelectric point.  

&nbsp;&nbsp;&nbsp;Anion exchangers can be classified as either weak or strong.  The charged group on a weak exchanger is a weak base that easily loses its charge at a high pH due to deprotonation.   On the other hand, strong anion exchangers are comprised of a charged group that is a strong base.  Strong anion exchangers are able to maintain their positive charges across a variable pH range while weak anion exchangers tend to lose their charge as the pH increases.  Examples of anion exchangers include the strong anion exchanger Q (quaternary resin), and the weak anion exchanger DEAE (diethylaminoethane).

&nbsp;&nbsp;&nbsp;Typically, the chromatography is performed using buffers at pH's between 7 and 10 and running a gradient from a solution containing just this buffer to a solution containing this buffer with 1M NaCl [http://www.proteinchemist.com/tutorial/iec.html (Res1)]. The salt (in solution) competes for binding to the stationary phase, thus releasing the protein from its bound state. Proteins separate because the amount of salt needed to compete varies with the external charge of the protein. Anion exchange chromatography have been successfully used to clean up of a crude slurry, separation of proteins from each other, concentrating a protein, and the removal of negatively charged endotoxin from protein preparations [http://www.proteinchemist.com/tutorial/iec.html  (Res1)].




	'''Ion exchange in practice.''' 
In order to elute an unknown protein via ion exchange, a gradient of different concentrations of salt with appropriate buffer is required to test at which salt concentration our desired protein is eluted. In the case of anion exchange, DEAE is used to pack our column. A buffer, such as Bis-tris, used to equilibrate, wash and elute. A syringe pump is commonly used to pump various buffer solution via the column. 
Before an ion exchange, target protein is first equilibrated in Bis-tris buffer. Some protein might precipitate in Bis-tris hence solubility could be a potential problem during ion exchange. The column is first equilibrated with buffer with no salt. One salt commonly used is sodium chloride. Buffers that contain gradually increasing salt concentration is prepared, such as 50mM NaCl, 100mM NaCl, 150mM NaCl, etc. At last, a 1M NaCl buffer is used to completely clean up the column. All buffer needs to have their pH adjusted because protein are pH sensitive. 
As the diagram as suggested, fractions of increasing salt concentration are collected. After all fractions are collected, samples are taken to run an SDS page. With the gel, we can tell at which salt concentration our protein was eluted, and how well the separation is. 
The testing scale does not require a lot of protein mixture. In large scale protein purification, salt containing buffer can start rather high as an effective wash. Samples will be taken from all fractions to track our purification. 

[[File:Ion exchange setup.jpg|thumb|Add caption here]]

===Cation Exchangers===
[[Image:Cations.png|thumb|right|400px|Figure 3. Cation Exchanger - [http://www.proteinchemist.com Proteinchemist.com]]]
&nbsp;&nbsp;&nbsp;Cation exchangers are named for their ability to attract cations or positively charged particles.  In this case, the resin of the chromatography system is negatively charged and proteins will bind if the buffer pH is less than the protein’s unique isoelcetric point.  

&nbsp;&nbsp;&nbsp;Just as with anion exchangers, cation exchangers can be classified as either weak or strong.  A weak cation exchanger is comprised of a weak acid that gradually loses its charge as the pH decreases, while a strong cation exchanger is comprised of a strong acid that is able to sustain its charge over a wide pH range.  Carboxymethal groups are used commonly as weak cation exchangers, while sulfopropyl groups are widely used as strong cation exchangers [http://www.rit.edu/~pac8612/webionex/website/html/ione0ni1.html (Res1)]<br>
<br>
<br>

===Designing a Cation Exchange Separation===
&nbsp;&nbsp;&nbsp;Selecting the correct resin and buffer is critical as it determines the binding properties of proteins to the resin. In cation exchange, the protein of interests needs to be positively charge to bind to the negatively charged stationary phase. Suppose we have a mixture of the 
following protein :

{| width="400" border="1"
|-
| <strong>Protein</strong>
| <strong>pI</strong>
| <strong>pH 4.8 cm</strong>
| <strong>pH 7.2 cm</strong>
| <strong>pH 8 cm</strong>
|-
| Carbonic Anhydrase
| 7.0
| +16.5
| -0.4
| -2.7
|-
| Carboxypeptidase B
| 6.2
| +12.0
| -3.3
| -6.3
|-
| Chymotrypsin
| 8.0
| +9.0
| +2.7
| 0.0
|-
| Lysozyme
| 9.8
| +14.1
| +7.9
| +6.9
|-
|}


*At pH 4.8 cm, all protein will bind to the cation exchanger, with carbonic anhydrase binding the strongest to the stationary. If elution is performed at this pH, the protein will elute in the order of chymotrypsin, carboxypeptidase B, lysozyme, and carbonic anhydrase. 

*At pH 7.2 cm, carbonic anhydrase and carboxypeptidase B will elute in the wash (before the gradient is initiated), followed by chymotrypsin, then lysozyme during the salt gradient.

*At pH 8 cm, only the lysozyme will bind to the stationary phase, the other three proteins will elute in the wash.

&nbsp;&nbsp;&nbsp;It is also possible to refine the salt concentration in solvents A and B so that the more weakly bound proteins will wash off the column in the wash, even if they are positively charged. For example, if you modify pH 4.8 cm to have 0.1 M NaCl in solvent A and 0.2 M NaCl in solvent B, you find that the most weakly bound protein (chymotrypsin) elutes in the wash, while the other three elute in the gradient.

==Resources==
#GE HealthCare [http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/4A555D526B88731AC1256E92003E865C?OpenDocument Ion Exchange Animation]
#Craig. P., Rochester Institute of Technology [http://www.rit.edu/~pac8612/webionex/website/html/ione0ni1.html Ion Exchange Chromatography]
#An online JAVA applet that simulates ion exchange chromatography can be found here: [http://www.rit.edu/~pac8612/webionex/website/html/simulate.html Ionex Simulation] (Directions on how to set up experiments and run them using the simulation can be found at the link)
#[http://courses.cm.utexas.edu/jrobertus/ch339k/overheads-1/ch5_ion-exchange.jpg Ion Exchange Image]
#Bio-Rad [http://www.bio-rad.com/B2B/BioRad/product/br_category.jsp?BV_SessionID=@@@@0655054085.1146799058@@@@&BV_EngineID=cccfaddhjklfdfkcfngcfkmdhkkdfll.0&categoryPath=%2fCatalogs%2fLife+Science+Research%2fChromatography+%7c+Protein+Purification%2fChromatography+Media%2fIon+Exchange&divName=Life+Science+Research&loggedIn=null&lang=English&country=null&catOID=-31119&isPA=false&catLevel=5&serviceLevel=null  Ion Exchange Chromatography & Products]
#Sigma-Aldrich [http://www.sigmaaldrich.com/Brands/Aldrich/Tech_Bulletins/AL_142/Sephadex_Products.html Ion-Exchange Ion-Exchange Resins and Related Polymeric Adsorbents]
#Rohm and Haas [http://www.rohmhaas.com/ionexchange/products.htm Ion Exchange Resin Products]

==References==
#The right step at the right time. Bio/Technology, 4, 954-958 (1986), Bonnerjera, J., Oh, S., Hoare, M., Dunhill, P.
#Amersham Biosciences [http://www.gelifesciences.com/file_source/GELS/Service%20and%20Support/Documents%20and%20Downloads/Handbooks/IEX%20Handbook.pdf Ion Exchange Chromatography Principles and Methods]*
#Vydac [http://www.seaviewsci.com/vydac/vydacpubs/vhpionex.pdf Principles and Applications of High-Performance Ion-Exchange Chromatography for Bioseparations] *
#Ellyn A. Daugherty [http://www.accessexcellence.org/AE/AEC/AEF/1994/daugherty_ion.html Using Ion Exchange Chromatography to Separate Proteins] *
.* Denotes Free Article

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