CHROMATOGRAPHY

CHROMATOGRAPHY

9.      CHROMATOGRAPHY

Chromatography is a physical & analytical tech. Which is used for separation of components of the mixture by using stationary & mobile phases. There are several types of chromatography which are categorised on several bases which are shown below :

9.1.    Column chromatography: -
Column chromatography is a type of chromatography in which the mixture of the sample is separated through the column made up of glass or steel. The mixture is separate through the column from top to bottom.

Property of column chromatography: -
Chromatogram: -
The chromatogram is the graphical representation of eluate volume with absorbance. The volume or time it takes for an analyte to come out from column known as retention volume or time.
Example:- in the chromatogram peak P and Q are separate peaks or R & S are overlap peaks, these overlap peaks are so known as the fused peak.


Retention time (tR) : 
Retention time is the total time in which solution passes through the column from injection of the sample to detection of the sample.
It is the amount of time solute takes to pass through the column. I.e. Time taken by solute to move from A to B. It is the characteristic time taken by a particular analyte to pass through the system I.e. from the column inlet to the detector under set conditions. 
tR of a compound is not fixed and influenced by many factors: flow rate, column length, temperate difference.     
Resolution:- 
The capacity of the column to separate the analyte from one another is known as resolution. The resolution also defines as the ratio of retention time between two peaks of analyte and average of the base of the peak which is explained by.

When R= 1 the Separation of two peaks is almost 100% and column with Rs more than 1.5 is considered good. The number of distribution events governs the ability of the column to separate the two analytes. In another word, distribution is directly proportional to the number of distribution events. In column chromatography, each thin plane of column Matrix participates in the distribution of a molecule. Assume height of a distribution plane is H and length of the column are L hence number (N) of distribution plane in a column is given by. 

Number of distribution plane a column is controlling two parameters:-
1)     As the number of distribution plane will go up, it will allow the analyte to travel longer period of time. Consequently, it will increase the distance between the two peaks.
2)     As the number of distribution plane will go up, it will require the width of the base of the peak, as a result, the peaks will be sharper.
As the shown number is increased, the peak width is decreasing. Hence, the number of distribution is an indirect way to measure the column efficiency. Higher N number is desirable for better separation. 

Eluent: 
It is the solvent used to carry the solute. (elute) the carrier of solute or analyte. It is solvent that will carry analytes. 
When the eluent flows through the column, the analyte get separated as per there distribution or partition coefficient and pass out at a different time as the eluate.
Basic components of column chromatography:
•    Stationary phase: Stationary phase is selected  on the basis of the type of analytes that are to be separated 
•    Column: Column is to be filled with stationary phase coated matrix or stationary phase is applied directly on the inner wall of the column.
•    Mobile phase: Mobile phase must be chosen competent to stationary phase and must be able to discriminate between analytes in the sample.
•    An Injection system: It is meant to deliver test samples to the top of the column from where the sample is allowed to flow through the column 
•    Detector: It detects the presence of the analyte in the eluate as it emerges out of the column. Detection is based on physical parameters like UV absorption or fluorescence. On the basis of information recorded by the detector, a recorder chart can be prepared in which peak represents separated analytes.
•    Fraction collector: fraction collector collects separated analytes
The stationary and Mobile phase
For efficient chromatographic separation, selection of stationary and mobile phase is very crucial because these features determine the effective separation of analytes as per difference in their distribution coefficients. There are certain ways to achieve this set up :
•    Ion exchange Equilibrium: This equilibrium exists between solid ion-exchanger (stationary) and a liquid electrolyte phase (mobile) 
•    Adsorption Equilibrium: This equilibrium exists between the stationary solid phase and the mobile liquid phase. E.g. Adsorption chromatography 
•    Partition Equilibrium: This equilibrium exists between the stationary liquid phase and a mobile liquid or gas phase. E.g. Gas-liquid chromatography 
•    Exclusion Equilibrium: It exists between a liquid phase trapped inside the pore of stationary phase and same mobile liquid phase. E.g. Gel filtration
•    Binding Equilibrium: This equilibrium exists between the stationary immobilized ligand and a mobile liquid phase. E.g. Affinity chromatography
Qualitative  Analysis 
Qualitative analysis is meant to check out the presence of the specific analyte in the test sample. This objective is carried out by :
•     Comparing the Retention time of the peaks in the chromatograph. Moreover, confirmation for the presence of the analyte can also be obtained by spiking a second test sample with a known amount of actual compound. The increased area with a single peak shows the presence of an analyte.
•    Nuclear magnetic resonance (NMR) spectrometer or mass spectrometer are used as the detector.
Quantitative Analysis
Quantitative analysis is performed for the purpose of identifying and confirming the presence and amount of a specific analyte in the test sample. 
Quantification of the analyte is based on peak area coupled with an appropriate calibration graph. In the chromatograph, the area of each peak is proportional to the amount of analyte because of which peak is produced. 
9.1.1.    Size exclusion chromatography (SEC) :- 
It is also known as gel filtration chromatography or molecular sieve chromatography or gel permeation. In this, the molecule gets separated on the basis of its size. In this, the sieving medium is a porous gel. It is named so because molecules get separated by the flow of solute and solvent through porous beads. These fine porous beads are packed into a chromatography column. The beads are composed of cross-linked dextran polymers (Sephadex), agarose (Sepharose), or polyacrylamide (bio-gel) cross-linked allydextron (Sephacryl). It is among the most gentle purification methods due to the lack of chemical interaction with resin.
Size exclusion chromatography (SEC) is quite different from other chromatographic techniques. It differs from gel permeation chromatography (GPC) which separates the organic polymers from non-aqueous systems. As well as from ion exchange chromatography, it does not depend on any chemical interaction with protein rather based on the physical property of the protein. In gel permeation, the organic solvent is used as mobile phase and gel permeation chromatography is a type of size exclusion chromatography (SEC). In this, the dry gel is used which first allow to swell in water and buffer under favourable condition.
When a mixture of proteins is poured into the column, some protein is small enough to enter the pores due to there low molecular weight and small size. So as a result, their rate of diffusion through the column is slowed down whereas larger proteins which are large in molecular weight aren't able to enter the beads and passes through bead pores uninterrupted without using bead volume.
So, total bead volume of the packed chromatography column, Vt will be 
Vt = Vo + Vi + Vg

Where Vo is volume outside porous beads, Vi is the volume inside the beads Vg is volume actually occupied by the bead material (Vg < 1% of Vt, so negligible)
9.1.1.1.    Experimental determination of chromatography parameters
When any molecule is able to enter pores in the beads then its distribution is given by the distribution coefficient, (KD) 
The behaviour of the molecule to be separated out is determined by 3 parameters.
(1)    Ve  =  elution volume  = Volume of eluent collected
(2)    Vi   =  Inner volume accessible to a solute molecule (bead volume)
(3)    Vo  = Void volume (Volume other than bead volume)

Where KD  =  distribution coefficient
here    KD  =  'O', when molecule utilise only void volume 
KD  =  1, when molecule utilise both void volume and bead volume (Vi and Vo).


When the molecular weight difference between the two proteins is not much, B start eluting out before A ends. This indicates that there is less difference in their molecular weight.


Resolution of this chromatography depends upon column length and volume of sample loaded.
Column length – All molecules need a particular time to interact with surrounding molecules. This property defines the relation of length with resolution. So longer the length of the column, better will be the resolution.
Sample volume – the Higher number of molecules starts a competition between molecules which gives much-overlapped peaks as a result. So, a specific limit of sample volume is defined which is 3-5% of the total column volume that is required to get separated and resolve overlapped peaks.


The graph plotted between, elution volume against the logarithm of molecular weight yields a straight line which indicates a gradual decrease in eluted volume of low molecular weight molecules.  
9.1.1.2.    Application –
Determination of native molecular weight of a protein using gel filtration chromatography
The molecular weight and size of a protein is related to the shape of the molecule and the relationship between molecular weight (M) and radius of gyration (Rg) is as follows- 

Rg \alpha Ma 

here “a” is a constant and it depends on the shape of the molecule, a=1 for Rod, a=0.5 for coils and a=0.33 for spherical molecules. The set of known molecular weight standard protein can be run on a gel filtration column and elution volume can be calculated from the chromatogram. A separate run with the analyte will give elution volume for the unknown sample. Using the following formula, Kd value for all standard protein and the test analyte can be calculated.


A plot of Kd versus log mol wt is given in graph, B and it will allow us to calculate the molecular weight of the unknown analyte.  


Oligomeric status of the protein:- Native molecular weight determination by gel filtration in conjugation with the SDS-PAGE can be used to determine the oligomeric status of the protein.  

Studying protein folding:- Protein is made up of the different types of amino acid residues linked by the peptide bond. As soon as the peptide chain comes out from the ribosome, it folds into the 3-D conformation directed by the amino acid sequence, external environment and other factors. Protein structure has a multilevel organization. i.e. Primary structure (sequence of the protein), secondary (α-helix, β-sheet and turn), tertiary and quaternary structure. When protein is incubated with the increasing concentration of denaturating agents (such as urea), it unfolds the native structure into the unfolded extended conformation following multiple stages. The different protein conformation forms during the unfolding pathway have the distinct hydrodynamic surface area and it can be used to follow protein folding-unfolding stages with the gel filtration chromatography. The details of the experimental setup are given in the fig. Protein is incubated with different concentration of urea (0-8M) for 8-10hrs at 37° C. A gel filtration column is equilibrated with the buffer containing urea (same as in incubation mixture) and the incubation mixture is analyzed. As the concentration of denaturating agent is increasing, protein will unfold with an increase in the hydrodynamic surface area. As a result, protein peak shifts towards the left. At the highest concentration of denaturant, protein unfolds completely and mostly appear in the void volume.  
Studying protein-ligand interaction-Gel filtration chromatography separates the molecules based on their size. Ligand binding to the protein induces conformational changes, resulting in the change in size or shape. In addition, the ligand is small in size whereas the protein-ligand complex is big and may appear at a distinct place in the column. In step 1, a gel filtration column is equilibrated with the buffer and elution profile of ligand is recorded. Now column is equilibrated with the buffer containing ligand molecule. As the concentration of ligand is increased, the protein binds ligand and form a larger complex with an increase in the hydrodynamic surface area. As a result, protein peak shifts towards the left. 


As the concentration of ligand will increase with a fixed amount of the protein, the free ligand will appear in the chromatogram. The protein amount the concentration at which free ligand appeared and the elution data can be used to calculate the stoichiometric ratio of ligand/protein and the equilibrium constant.  


Desalting- Desalting or removal of the small molecule from the protein is important for activity assay and other downstream processes. A gel filtration column is equilibrated with the buffer or water and then the sample for desalting is loaded. After the run, the protein and salt are eluted separately as the peak.

9.1.2.    Hydrophobic Interaction Chromatography (HIC)
This technique uses the hydrophobic nature of proteins to purify them from a mixture. This technique was first described by Shepard & Tiselius (1949) and used the term “salting-out chromatography”. Later, Shaltiel introduced the term “hydrophobic chromatography”. It's principle is complementary to size exclusion chromatography and & Ion-exchange chromatography.
The mixture of proteins is passed over a chromatographic column which is packed with a support matrix to which hydrophobic groups are covalently linked. Phenyl Sepharose, agarose is most commonly used support matrix to which hydrophobic groups are attached.


Butyl is the shortest group to behave as hydrophobic and phenyl have the strongest hydrophobicity. Prior to interacting with support protein mixture is treated with high salt concentrations because hydrophobic groups have a tendency to bury themselves internally in the protein 3D structure but some may be exposed. So salts can be used to expose these hydrophobic groups. These exposed hydrophobic group can interact with support and can precipitate or crystallized proteins out of solution. The most hydrophobic protein retained the longest on the column. More the hydrophobicity, more it will stay in the column. 
Likewise, for the elution of proteins from interaction with phenyl groups can be done by lowering the salt concentration or by adding organic solvents such as polyethylene glycol to the elution fluid. Globular proteins are highly folded and are more tolerant of high salt concentrations so hydrophobic groups are remain buried in them and they are not able to interact with the surrounding aqueous solvent.


9.1.2.1.    Factors affecting interaction with matrix  
Ligand type---Straight-chain alkyl ligands are more hydrophobic and can interact with both aromatic and hydrophobic molecules. So as the alkyl chain length increases, the binding capacity of protein increases. 
Type of base matrix --- cross-linked agarose and synthetic copolymer materials are used.
Type and concentration of salt---Na or K, ammonium sulfates are highly useful for salting out mechanism so they are much able to promote ligand-protein interactions.
pH---increase in pH levels, weak hydrophobic interactions. It is due to increased titration of charged groups leading to an increase in hydrophobicity of the proteins. The decrease in pH leads to an apparent increase in hydrophobic interaction. A good starting pH is 7.0, irrespective of the component’s isoelectric point. 
Temperature---performed at room temperature or at 4°C. A higher temperature can affect binding strength and selectivity.
Additives-----Alcohols and detergents (non-polar parts) are used as additives which can compete with protein for interaction with support and can displace proteins.
9.1.2.2.    Advantages of HIC 
The large volume of sample can be loaded and Samples with high ionic strength can be used. This technique is used before gel filtration, ion-exchange and affinity chromatography techniques for sample purification. An ideal HIC process has the ability to good mass recovery after the purification process and retention of biological activity of all the denatured protein sample.
9.1.2.3.    Applications polishing monoclonal antibodies -  HIC ligands with different hydrophobicities are used and a range of resin is prepared so that offers a range for binding of the antibody according to its hydrophobicity.


9.1.2.4.    Aggregate Removal of HIC is often used to remove aggregates generated in protein purification steps for mAbs. These impurities have more hydrophobicity than the native protein so they form aggregation by hydrophobic interactions between its molecules and also have chemical properties very similar to the target. Therefore, they bind with the support and native proteins with relatively low hydrophobicity eluted out from the column. Impurities remain attached to the matrix.
9.1.2.5.    Purification tool for biomedical applications like vaccines, therapeutic proteins, plasmids and antibodies and also for high-throughput studies as proteomics and protein interactions.
9.1.3.    Reverse phase chromatography this method employs harsher conditions than HIC that can lead to denaturation. The basis of reverse phase chromatography is the hydrophobic interaction between proteins/peptides and support which contains alkyl or aromatic hydrocarbon ligands. Use of hydrophobic support is required as a stationary phase which is rather reverse from the normal chromatography.
It employs a polar (aqueous) mobile phase. That means Hydrophobic molecule in the polar mobile phase. Initially, silica was used as the stationary phase to which hydrophobic ligands were linked. But this support has a disadvantage that it is unstable at extreme pH change. Polystyrene is much better than silica support as a base matrix for cation exchanger is used for separation of amino acids. The greatest advantage of polystyrene is its chemical stability in acidic or basic conditions.

All hydrophobic molecules are buried in the cage of water molecules and hydrophobic force is a tendency of the water molecule to make a bigger cage. So, if any molecule surrounded by water binds with ligand then water molecules get displaced and hydrophobic molecule gets interacted. Displacement of the water molecule makes the interaction more favourable.
C-18 is the most hydrophobic ligand and is used for small peptide purification while the C-2 and C-4 ligands are more suitable for proteins. This technique is useful to check the purity and analysis by either micro purification of protein fragments for sequencing or scale purification of recombinant protein products.
9.1.3.1.    Comparison between HIC and RPC


9.1.4.    Affinity chromatography
In Affinity chromatography, some proteins have the ability to bind ligands tightly but non-covalently. So this technique uses this property of proteins to separate it from the mixture. It relies on the interaction between protein and immobilized ligands. For example, the antibodies in a serum sample are specific for particular epitope and can be isolated by the use of affinity chromatography.
The sample of protein is passed through a column which contains specific groups which have a higher affinity for a specific group of proteins and gradually they shows a decrease in the elution volume. For the elution of attached proteins, an exogenous or free ligand is added which compete with covalently linked ligand for the binding site of the protein and another method is to chemically change the solution by changing pH which reduces protein-ligand binding.
Several ligands and their respective receptors are used for affinity-based chromatographic separations like histidine tags, which are attached at any protein on either N or C terminal of protein, shows its high affinity for ligand, imido acetic acid just like glutathione-S-transferase protein is able to bind with the ligand glutathione which is immobilized on agarose.
Steps:-
Step 1 - Preparation of matrix:- Many types of polymers like agarose, cellulose, Sephadex are used. Now several target molecules are immobilized on a matrix like glutathione. Mostly substrates which are used for support should be in the sample solution of the target molecule, chemical groups should be easily modified for ligand attachment and mechanical and chemical stability.
Step 2 - The sample is passed through the matrix. As the molecules found their target molecule, they bind with them noncovalently and does not move further and other molecules which do not have their target molecule pass through matrix unimpeded (without hindrance).
Step 3 - Elution – It is the process to dissociate attached molecules from the resins. It can be done in several ways which are : 
1.    pH elution by altering pH of any solution the degree of ionization of the charged groups of the ligand or bound protein is changed and this alteration of charge may affect the binding sites of that molecule and reduce their affinity towards their respective molecules. It can also affect the conformation of ligands.
2.    Ionic strength elution: The exact mechanism for elution by changes in ionic strength will depend upon the specific interaction between the ligand and the target protein. This is a mild elution using a buffer with increased ionic strength (usually NaCl) Enzymes usually elute at a concentration of 1 M NaCl or less.


3.    Competitive elution: Several chemicals are used which competes with the ligands for binding with target molecules so this competition reduces the affinity of ligands for the target and elute them.
4.    Chaotropic eluents: These are the hydrophilic chemicals which can reduce the interaction between molecules. eg. : – Ethanol, Butanol, Propanol, Urea etc. these are used as the last treatment for elution if all other treatments are failed as these chemicals can denature proteins. Therefore, affects its stability.
Step 4 - Dialysis- The eluate is dialyzed to remove the reagent used for the elution.


Lectin affinity chromatography is a form of affinity chromatography in which lectins are used as a target molecule which binds to carbohydrate molecules.
9.1.4.1.    Use 
1.    Purify and concentrate a substance from a mixture into a buffering solution.
2.    Identification of particular target molecule of any biological compounds. 
3.    Purification of enzymes. 
4.    Purification of Recombinant proteins 
5.    Antibody purification -
1.    Precipitation with ammonium sulfate.
2.    Affinity purification with immobilized antigen.
Applications of affinity chromatography
Affinity chromatography is further divided into the different types based on the nature of receptor present on the matrix to binds tag present on the analyte molecule. Different types of affinity chromatography are-  
Bio-affinity chromatography- In this type of affinity chromatography, biomolecules are used as receptor present on the matrix and it exploits the biological affinity phenomenon such as antibody-antigen. In addition, enzyme-substrate or enzyme inhibitor has also belonged to this class. Ex. GST-Glutathione.
Pseudo-affinity chromatography-In this affinity chromatography, a non-biological molecule is used as the receptor on the matrix to exploit the separation and purification of biomolecules. There are two specific examples of this class-  
A.     Dye-affinity chromatography-In this method, the matrix is coupled to the reactive dye and the matrix-bound dye has specificity towards a particular enzyme. For ex. Cibacron Blue F3G-A dye coupled to the dextran matrix has a strong affinity towards dehydrogenases. 
B.     Metal-affinity chromatography-In this method, transition metals such as Fe2+, Ni2+ or Zn2+ is coupled to the matrix and the matrix-bound metal form the multidentate complex with protein containing the poly-his tag (6x His). The affinity of the protein for matrix-bound metal is different and these differences are been exploited in metal affinity chromatography to purify the protein.    
Covalent chromatography- This is a different type of chromatography technique where binding of the analyte to the matrix is not reversible as it involves the formation of a covalent bond between functional group present on matrix and analyte. Thiol group (SH) present on neighbouring residues of protein forms disulphide bond after oxidation and under reducing environment. This disulphide is reversibly broken back to a free thiol group. The matrix in covalent chromatography has immobilized thio group which forms a covalent linkage with the free thiol group-containing protein present in the mixture. After a washing step to remove non-specifically bound protein, a mobile phase containing a compound with reducing thio group is passed to elute the bounded protein. The thio group-containing compound present in mobile phase breaks the disulphide bond between protein and matrix thio group to release the protein in the mobile phase.  


Applications of Affinity Chromatography 
1.      Purification of biomolecules GST Based Purification-
Glutathione S-transferase (GST) utilizes glutathione as a substrate to catalyze conjugation reactions for xenobiotic detoxification purposes. The recombinant fusion protein contains GST as a tag that is purified with glutathione coupled matrix. GST fusion protein is produced by the recombining protein of interest with the GST coding sequence present in the expression vector (either before or after coding sequence of the protein of interest). It is transformed, over-expressed and the bacterial lysate containing fusion protein is purified, using affinity column. The sample is loaded on the column previously equilibrated with the buffer containing high salt (0.5M NaCl). Unbound protein is washed with the equilibration buffer and then the fusion protein is eluted with different concentration of glutathione dissolved in the equilibration buffer. Purified fusion can be treated with the thrombin to remove the GST tag from the protein of interest. The mixture containing free GST tag and the protein can be purified using the affinity column again as the tag will bind to the matrix but protein will come out in the unbound fraction. 


2.     Protein-Protein interaction-Protein-protein interaction can be studied through multiple techniques or approaches. Affinity column also can be used as a tool to study or isolate interacting partner of a particular protein. A schematic figure to depict the steps involved in the studying protein-protein interaction. In this approach, the matrix is incubated with the pure protein-1 and then washed to ensure tight binding. All other sites on the bead are blocked with a non-specific protein such as BSA or an unrelated cell lysate. Now, cell lysate or the pure protein-2 is passed to the protein-1 containing beads, followed by washing with the buffer to remove unbounded proteins. Now, the protein-1 is eluted from the matrix either by adding a high concentration of ligand or with the denaturating condition. Now the eluted protein is analyzed in the SDS-PAGE or SDS-PAGE followed by the western blotting to detect protein 1 or protein 2. As a control, cell lysate or protein-2 is also added to the matrix without protein-1 to rule out the possibility of protein-2 binding directly to the matrix.

3.     Enzymatic Assay-Affinity chromatography can be used to perform enzymatic assay such as protease assay. In this assay, peptides with different amino acid sequence bound with the terminal residue to the affinity bead and incubated with the protease for an optimal time. The enzyme acts on the attached peptide and releases, the free portion into the supernatant. The supernatant is recovered from the reaction mixture and can be analyzed in a MALDI-tof to deduce the amino acid sequence from the molecular weight. Analysis of a set of reaction may allow to predict the protease recognition and cutting site

4.     Clinical diagnosis-Receptor present on the matrix provides a unique tool to isolate,  detect and characterize biomolecules from the crude mixture.  For example, a matrix containing boronic acid is used to separate and quantify glycosylated haemoglobin from diabetic patients blood. Ribonucleoside in patient urine can be identified by an affinity matrix containing boronic acid followed by the reverse phase chromatography. 
5.    Immuno-purification- The avidin-biotin system is used to capture and isolate cytokines from immune cells. Biotinylation of antibodies allows immobilization of antibodies in the correct orientation on the streptomycin coated glass beads. Lymphocyte lysate is passed to the column packed with the glass beads containing antibodies that bind cytokines. The cytokines are eluted by flowing buffer of decreasing pH or by chaotropic ions. The antibodies remain bound to the column due to strong affinity between avidin-biotin which is resistant to these chemical treatments. 


9.1.4.2.           Table showing various functional group use in affinity chromatography and its application.

Ion exchange chromatography: -
Ion exchange chromatography is high-resolution chromatography technique that purifies protein on the basis of charge present on it.
Principal:- 
In ion exchange chromatography analyte molecules are distributed on the basis of charges and their affinity for opposite charge present on matrix competitive counter Ion Exchange with Matrix-bound analyte for elution. The interaction between analyte and matrix is determined by ionic strength, net charge and pH of the buffer. 
For Example:- Mixture of analyte (A+, B, C-, D-2) loaded in the negatively charged matrix containing column. Neutral nature molecule and negative charge molecule elute first through the column whereas positively charged molecule bind with Matrix charge for elution of positive charge from Matrix, the high concentration of counter ion is needed. 

The Matrix used in Ion exchange chromatography is present in the ionized form with the reversibly bound ion to the matrix. Two type of Ion exchange chromatography is available.
1)     anion exchange chromatography.
2)     cation exchange chromatography.
1)     Anion exchange chromatography:- 
In anion exchange chromatography, the positively charged matrix shows an affinity for the negative charge. The binding of the negatively charged matrix replaces bounded anion from the matrix. For elution of negative charge, the strong anion (Cl-) is used as mobile phase. For replacement of aligning with the matrix.
2.    Cation exchange chromatography:-
In cation exchange chromatography, the negatively charged matrix shows an affinity for positive charge. The binding of positive charge molecule replaces bounded cation from the matrix. For elution of positive charge, strong cation (Na+) is used as the mobile phase for replacement of cation with the matrix.


Isoelectric point and charge on protein:-  
Protein is a polymer made up of Amino acid with an ionizable side chain. The variation on the net charge is due to variation in pH. Isoelectric points that pH at which net charge of the amino acid is zero. Above the pH value, the net charge of protein show positive nature and below the pH value, it shows negative nature.
Choice of Ion exchange column matrix:- 
In ion exchange chromatography, selection of Ion exchange column is very important for purification of analytes.
There are some criteria for selection of matrix-
pI value and net charge:- To calculate net charge at particular pH, we need pi value of protein. Below the pI value, we use cation exchange chromatography and for above the pI value, we use anion exchange chromatography. 

Structural stability:- Structure of a protein is maintained by electrostatic and Vander wall interaction between charged amino acid, hydrophobic interaction between the hydrophobic side chain of amino acid and hydrogen bonding between the polar amino acid. As a result, protein structure is stable in a narrow range around its pI value. A large deviation from it may affect its 3D structure. 
Enzyme activity:- There is a very short range of PH in which enzyme shows its activity. The range of PH to be chosen must be favourable for the protein.
Application:- 
Protein Purification - protein purification can be achieved with help of Ion exchange chromatography because. All protein have particular pi value at which its net charge is zero. If pi value less than pH for the given protein that protein becomes negatively charged (an anion) and if PH value is greater than for given protein, it becomes positively charged (cation).
Ion exchange chromatography occurs due to electrostatic attraction between buffer is all discharge protein and oppositely charged by insight on a solid Ion exchange absorbent. Usually consists of spherical forest beat with exchange group (functional group).
Ion exchange chromatography occurs due to electrostatic attraction between buffer-dissolved charged proteins and oppositely charged binding sites on a solid ion exchange adsorbent. An ion exchange adsorbent (also called media, resin, gel, or matrix) usually consists of spherical porous inert beads with charged groups (functional groups) densely grafted onto the beads surfaces. The charges of functional groups are neutralized by free counter-ions.
steps for ion exchange chromatographic purification
The protein mixture is transferred into low ionic strength buffer in the mobile phase.
Ion exchange adsorbent (stationary phase) is packed into a column and the column is pre-equilibrated with the buffer of identical pH and similar ionic strength as protein mixture (preferably the same buffer as protein mixture).
The mechanism involved in ion exchange :
The protein mixture is applied to the column. Proteins charged oppositely to ion-exchange media are temporarily retained in the column. All other proteins simply pass through the column and are collected during this step.
Retained proteins are eluted from the column by applying a modified buffer. Elution is most commonly achieved by gradually increasing the ionic strength of the buffer via salt gradient, and proteins are eluted in order of increasing their net charges. In specific cases, the elution can be accomplished by (a) pH change and (b) affinity methods.
Ion exchange chromatography can provide high-resolution separation for proteins with the same sign but different total net charge. Due to the high capacity of most ion-exchangers, the technique can also be used for capture of a mixture of same-sign charged proteins from large-volume diluted samples, the proteins are then eluted in the considerably decreased sample volume.

Application: During the application of the protein mixture onto the stationary phase, proteins adsorb from the solution to oppositely charged ion-exchanger media by displacing buffer ions that originally balance charges on the functional groups. The adsorption is reversible and occurs in continuous competition for the binding sites between same-sign charged proteins and the buffer ions. In low ionic strength buffers, the concentration of competing for buffer ions is low and proteins spend most of their time adsorbed to binding sites on the stationary phase. However, even strongly bound proteins still spend some time in the solution and therefore they continue to slowly move down the column with buffer flow. For example, if a given medium-bound protein spends 95% of the time adsorbed to the stationary phase, this protein will move down the column at 5% speed of the buffer have been applied. Proteins bearing smaller electric charges move down the column faster than proteins bearing larger electric charges.
Elution with salt gradient:- Addition of salt increases the number of ions competing with proteins for functional groups on the stationary phase. Proteins spend more time in the solution, the rate of their movement down the column increases dramatically, and proteins begin to elute from the column, usually in the order of increasing charge. Most proteins are eluted at NaCl concentrations < 1M.
Elution by pH change:-  Change of pH in the column can be aimed to decrease the net absolute value of the charges of adsorbed proteins, decrease their attraction to the stationary phase and accelerate the elution. In practice, pH changes in the column are difficult to control, as they do not reliably correspond to pH changes of the applied eluting buffer. This happens because of the buffering power of proteins adsorbed to the column and for weak ion exchangers (see below), buffering power of the adsorbent functional groups themselves. Resolution of proteins by pH elution is achieved in a separate technique called “Chromatofocusing.”
Protein-DNA interaction-Ion-exchange column is used as a tool to study the interaction between DNA and a particular protein. DNA is negatively charged and has a strong affinity towards anion exchange chromatography. A schematic figure to depict the steps involved in DNA-protein interaction is given in Figure. In this approach, the anion exchange matrix is incubated with the DNA and allowed it to bind tightly. Excess DNA is washed from the column. Now the pure protein is passed through the DNA bound beads, followed by washing with the buffer to remove unbounded proteins. Now the DNA is eluted from the matrix either by adding a high salt concentration or with the denaturating condition. Now the fractions are tested for the presence of DNA and protein. The eluted protein is analyzed in the SDS-PAGE and DNA is in agarose. As a control, protein is also added to the matrix without DNA to rule out the possibility of protein binding directly to the matrix. If protein will have an affinity towards DNA, they both will comes out from the column at the same time and should give similar pattern in the elution profile. It could be possible that high salt may break interaction between DNA and protein, in such situation protein will come out first followed by DNA. Besides this ion-exchange chromatography approach still be able to answer whether the DNA-protein is interacting with each other or not. 

3.     Softening of water-
Groundwater has several metals such as Ca2+, Mg2+ and other cationic metals. Due to the presence of the metal, hard water creates a problem in industrial settings. Ion-exchange chromatography is used to remove the metals present in the water through an exchange of matrix-bound Na+. Calcium or magnesium present in the hard water has more affinity towards the matrix and it replaces with matrix-bound sodium ions. The schematic presentation of water softening is given in Figure. A cation exchanger matrix with bound sodium is packed in the column and the hard water containing calcium, magnesium is passed through the column. In this process, calcium present in the solution preferentially migrates from the solution to the matrix whereas sodium ion present on the matrix migrate into the solution. The matrix can be used for softening of the water until it has bound sodium ions. 

Once sodium ions are exhausted, the matrix can be regenerated by flowing a solution of sodium chloride or sodium hydroxide. The calcium/magnesium bound to the matrix comes out in the solution and can be dumped into the sewage.   


4.    Protein kinase assay- 
The protein kinase is the class of enzyme responsible for the transfer of phosphate group on the substrate molecule. In the protein kinase assay, a radioactive substrate (preferable a radioactivity on carbon) was incubated with the enzyme protein kinase, MgCl2 and non-radioactive ATP. A negative control is also been included where an enzyme protein kinase is absent from the assay mixture. The reaction mixture from negative control and experimental will be loaded on two separate cation exchange chromatography columns to bind unphosphorylated substrate from the reaction mixture whereas the phosphorylated radioactive substrate is present in the flow through. The radioactive count of the flow through was measured using a liquid scintillation reagent.


5.    Purification of rare earth metals from nuclear waste- 
Ion-exchange matrix is used to isolate and purify rare earth metals such as uranium or plutonium. The first process to isolate uranium in large quantities was developed by Frank spending. Ion-exchange beads are also found suitable to recover uranium from the water coming out of the nuclear power plant. Uranium binds to the matrix through the ion-exchange process. The uranium bound bead is sent to the processing unit where uranium is isolated from the beads to form ‘yellow cake’ and stored in the drum for further processing. The ion-exchange beads can be reused in the ion-exchange facility.


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