AFFINITY CHROMATOGRAPHY

ANALYTIAL CHEMISTRY

CHROMATOGRAPHY
Chromatography is a powerful separation method that finds applications to All branches of science. Chromatography was invented and named by the Russian botanist Mikhail Tswett at the beginning of 20th century. Chromatography encompasses a diverse and important group of methods the permit the scientist to separate closely related components of the complex mixtures. In all chromatographic separations the sample is transported in mobile phase, which may be gas a liquid or supercritical fluid. This mobile phase is then forced through an immiscible stationary phase, which is fixed in palaces in a column of or on a solid surface. The two phase are chosen so that the components of the sample distribute e themselves between the mobile and stationary phase to varying degrees. Those components the t are strongly retained by the stationary phase move only slowly with the flow of mobile phase. In contrast, components that are weakly held by the stationary phase travel rapidly. As a consequence of these differences in mobility, sample components separate in to discrete bands, of zones, that can be analyzed qualitatively and quantitatively.

CLASSIFICATION OF CHROMATOGRAPHY

Depending upon the mobile phase and stationary phase-: 1. Liquid Chromatography. 2. Gas Chromatography. 3. Supercritical fluid chromatography.

Affinity chromatography Definition: Affinity chromatography is a liquid chromatographic technique

that uses a well defined binding agent as the stationary phase for the selective purification or analyses of sample components. The retention of solutes in this method is based on the specific reversible interactions as that occur in biological systems, such as binding of enzyme with a substrate or an antibody with an antigen. These interaction are used is affinity chromatography by immobilizing or adsorbing one of a pair of interacting molecules on a solid support and placing this modified support into a column. The immobilized or adsorbed molecule that is used as the specific binding agent is known as the affinity ligand. The presence of this ligand results in a stationary phase that has the ability to bind to the complementary molecules in samples that are applied to the affinity column.

Theory :

The

most commo method used for performing affinity

chromography is a step wise elution scheme as shown in fig below:

Step 1. Loading affinity column.

Sample containing the compound of interest is injected onto the affinity column in the presence of a mobile phase that has the right pH, ionic strength and solvent composition for the solute-ligand binding. This solvent which

represents the weak mobile phase of the affinity column is called the application buffer. As the sample passes through the column in the application buffer, compounds which are complementary to the affinity ligand will bind. Step 2:

However owing to the high selectivity of this interaction other solutes in the sample will tend in elute from the column, non-retained. After all non retained components have been washed from the column the retained solutes are then eluted by applying a solvent that displaces them from the column or that promotes dissociation of the solute-ligand complex. This solvent, which represents the strong mobile phase for the column is known as the elution buffer. As the solutes of interest elute from the column, they are either measured directly or collected for the later use. Step 3:

The application buffer is then reapplied to the system and the column is allowed to regenerate prior to the next sample injection. Factors like strength of the solute-ligand interaction, the amount of immobilized ligand present in the column and the kinetics of solute-ligand association and dissociation determine the retention and elution of a compound in an affinity column.

Basic components of Affinity System :_

1. Affinity Ligands:- The key factors in determining the success of any affinity separation is the types of ligand that is used within the column. Many of these ligands are of biological origin, but a wide range of natural & synthetic molecules of non biological origin can be used. The ligands are of 2 types. A. High-Specificity ligands B. General or group specific ligands. A. High-Specificity ligands:- It refers to compounds which bind in only one or a few closely related molecules. This type of ligand is used to analyse to purify a specific solute. Typical high-specificity ligands include antibodies, substrates or inhibitors and single-stranded nuclic acids (for the retention of a complementary sequence).Most high-specificity ligands tend to be biological compounds. These ligands also tned a have relatively large association constants for solutes and are generally eluted by a step gradient. B. General or group specific ligands:- These are the compounds that bind to a family or class of related molecules. These ligands are used to isolated a class od structurally similar solutes. General ligands can be of either proteins A & G, lectins, boronates, triazine dyes, immobilized metal chelates.

2. Support Materials- Another important factor to cons ides in affinity chromatography is the material used to hold the ligand within the column. The support material should have low non-specific binding for sample components but should be carry to modify for ligand attachment. This material should be stable under the flow rate, pressure and solvent conditions to be employed in the analysis of purification of samples. The support should be reading available and simple to use in method development. Depending on the type of support material, affinity chromatography can be characterised as:A. Low performance technique B. High performance technique A. Low performance technique:- in this, the support is usually a large diameter, non rigid gel. The low back pressure of these supports means that they can often be operated under gravity flow or with a peristaltic pump. This makes these gels, relatively simple & inexpensive to use for affinity purifications. Disadvantages of these materials include their slow mass transfer properties and their limited stability at high flow rates and pressures. B. High performance technique:-The support consists of small, rigid particles capable of withstanding the high flow-rates/pressures media. The mechanical stability and efficiency of these supports allows them to be used with standard HPCL equipment.

3. Immobilization

Methods:- a third item to consider in using affinity

chromatography is the way in which the ligand is attached to the solid support, or the immobilization method. For a protein or peptide, this generally involves coupling the molecule through free amine, carboxylic acid or sulfhyary residues present in its structures. Immobilization of a ligand through other functional sites

All immobilization methods involve at least 2 steps 1. An activation step:- in which the support is converted to a form that can be chemically attached to the ligand. 2. A coupling step:- in which the affinity ligand is attached to the activated support. The choice of immobilization method is important since it can affect the actual or apparent activity of the final affinity column. If the correct procedure is not used, a decrease in ligand activity can result from multisite attachment, improper orientation of steric hindrance.

Multisite attachment:-

Refers to the coupling of a ligand to the

support through more than one functional group which can lead to distortion and denaturation of the ligands active region

Improper attachment:- lead to a ;less in ligand activity but can be

avoided by coupling the ligand through group that are distant from its active region.

Steric hindrance:-Refers

to the loss of ligand activity due to the

presence of a nearby support or neighbouring ligand molecules.

Illustration of multisite attachment improper orientation & steric hindrance effects in affinity chromatography

MODS OF ELUTION IN AFFINITY CHROMATOGRAPHY

The elution conditions are chosen to promote fast removal of solute from the column. The elution buffer used in affinity chromatography can be either buffer that produce weak solute-ligand binding or a solvent that decreases the extent of this binding by using a competing agent that displaces solute from the column. These two approaches ate known as non-specific elution & bio specific elution.

A. Bio specific elution is the gentler of these two elution methods since it is carried out under essentially the same solvent conditions. Bio specific elution may be performed either by adding an agent to the eluting solvent that competes with the ligand for solute or by adding an agent that competes with solutes for ligand binding sites. In both cases, retained solutes are eventually eluted form the column by displacement & mass action. The main advantage of bio specific elution is its ability to gently remove analyte from the column. The main disadvantage are the slow elution times & broad solute peaks that ate typically produced by this approach other limitations include the need to remove the competing agent that does not produce a large background signal under the conditions used for analytic detection. a. In non-specific elution, the solute is eluted by changing the column conditions in order to weaken the interactions between any retained compounds & the immobilized ligands. This can be done by changing the pH, ionic strength or polarity of the mobile phase. The addition of denaturing agents to the running buffer can also be used. This results in an alteration in the structure of the solids or ligands, leading to a lower association equilibrium constant & lower solute retention. Non specific elution tends to be much faster than Bio specific elution in removing analytes from affinity columns. This results in sharper peaks which in turn produces lower limits of detection & shorter analysis time.

APLLICATIONS

1) Affinity purification of albumin and macroglobulin contamination Affinity purification is a helpful tool for cleaning up and removing excess albumin and 2- macroglobulin contamination from samples since these components can mask or interfere with subsequent steps of analysis (e.g. mass spectrometry and immunoprecipitation). One purification method which can be used to remove these contaminants either before or after other purification steps is Blue sepharose affinity chromatography. In this method, the dye ligand is covalently coupled to sepharose via a chlorotriazine ring. Albumin binds in a nonspecific manner by electrostatic or hydrophobic interactions with the aromatic anionic ligand. The most commonly used dye is Cibacron blue F-3-GA which can be immobilized onto Affinity Chromatography 22 sepharose to create an affinity column. This dye is capable of removing over 90% of albumin in the sample. 2) Protein A, G, and L purification Proteins A, G, and L are native or recombinant proteins of microbial origin which bind specifically to immunoglobulin including immunoglobulin G (IgG). IgG represents 80% of serum immunoglobulin. Native and recombinant protein A can be cloned in Staphylococcus aureus. Recombinant protein G (cell surface protein) is cloned in Streptococcus while recombinant protein L is cloned from Peptostreptococcus magnus. Both protein A and G specifically bind the Fc region of IgG while protein L binds to the kappa light chains of IgG. The most popular matrixes or supports for affinity applications which utilize protein A, G, or L is beaded agarose (e.g. Sepharose CL-4B; agarose crosslinked with 2,3- dibromopropanol and desulphated by alkaline hydrolysis under reductive conditions), polyacrylamide, and magnetic beads (Grodzki & Berenstein, 2010; Hober et al, 2007; Katohet al, 2007; Tyutyulkova & Paul, 1995). All three proteins bind extensively with the IgG subclass. In general, protein A is more suitable for cat, dog, rabbit and pg IgG whereas protein G is generally more preferable when purifying mouse or human IgG. A combination of protein A and G is also applicable for purifying a wide range of mammalian IgG samples. Since protein L binds to the kappa light chain of immunoglobulin and these light chains exist in other immunoglobulin (i.e IgG, IgM, IgA, and IgE), protein L is suitable for the purification of different classes of antibodies. The binding characteristics of antibody binding proteins (A, G and L) to a variety immunoglobulin species is summarized in IgGs from most species bind to

protein A and G near physiological pH and ionic strength. To elute purified immunoglobulins from protein G sepharose, the pH should be less than 2.7. S 3) Biotin and biotinylated molecules purification If a biotin tag can be incorporated into a biomolecule, it can be used to purify the biomolecule using a streptavidin or avidin affinity support. One way is to insert a Affinity Chromatography: Principles and Applications biotinylation sequence into a recombinant protein. Biotin protein ligase can then be used to add biotin in a post-translational modification step (Cronan & Reed, 2000). Biotin, also known as vitamin H or vitamin B7, is a relatively small cofactor present in cells. In affinity chromatography it is often used an affinity tag due to its very strong interactions with avidin and streptavidin. One advantage of using biotin as an affinity tag is that it has a minimal effect on the activity of a large biomolecule due to its small size (244 Da). Streptavidin is a large protein (60 kDa) that can be obtained from Streptomyces avidinii and bind biotin with an affinity constant of 1013 M1. Avidin is a slightly larger glycoprotein (66kDa) with slightly stronger binding to biotin (1015 M1). Both avidin and streptavidin have four subunits that can each bind one biotin molecule. To purify biotinylated biomolecules, streptavidin is immobilized onto a support material and used to extract the biotinylated molecules out of solution. Both avidin and streptavidin may be immobilized using aminereactive coupling chemistries. In addition, avidin can also be mmobilized via its carbohydrate residues. Due to the strong interaction between biotin and (strept)avidin, harsh elution conditions arerequired to disrupt the binding. For example, 6 M guanidine hydrochloride at pH 1.5 is commonly used to elute the bound biotinylated biomolecule. This prevents the recovery of most proteins in their active form. To overcome this difficulty, modified (strept)avidin or modified biotin may be used to create a lower affinity interaction. In one study chemically modified avidinhad relatively strong binding (>109 M1), but was also able to completely release biotinylated molecules at pH 10 (Morag et al, 1996). In addition, at any pH between 4and 10, a 0.6 mM biotin solution could be used to displace and elute the biotinylated molecules. Biotin is also used in isotopically coded affinity tags (ICATs) which can be used to compare the protein content in two different samples (Bottari et al, 2004). The ICAT consists of twolabels, one which contains deuterium (heavy) and one which contains only hydrogen (light). The two labels (light and heavy) are added separately to the cell lysates being compared. Since the reagent contains a thiol-specific reactive group, it will covalently bind free

cysteines on proteins. The labeled lysates are combined, digested with trypsin, and then isolated on a streptavidin column. After a second separation step, the labeled proteins are analyzed using mass spectrometry. The change in protein expression between the two cell lysates can then be quantified and related to the different conditions applied to the two sets of cell lysates.

Refferances
1. Principles of Instrumental Anakysis fifth edition by Skoog,Holler,Nieman. 2. Chromatography and electrophoresis techniques Smith Menemann interscience 1960 3. Extraction Chromatography T.Braun , G. Ghersene, Elsevier Publications 1978 4. Practical H.P.L.C. chromatography forth edition by Veronika R. Mayer. John Wiley & sons. 5. Analytical concepts fourth edition by R.A.Day & A.A. Underwood . 6. Chromatography concepts & contrasts by James M. Miller.

AFFINITY CHROMATOGRAPHY