CHROMATOGRAPHY

BY: RAHUL ANDHARIA (MSIWM001)

Enzyme Purification: The process in which enzymes are purified to obtain pure biological catalysts to study their nature and further using it in industries and in applied sciences to develop various                by-products.

Chromatography: It is a technique in which components are identified, separated and purified from a mixture for qualitative and quantitative analysis. Based on Polarity, net charge and hydrophobic interactions, enzymes are separated by using chromatography. In the year 1903, Chromatography technique was first used for colourful separation of plant pigments through a calcium carbonate column by Mikhail Tswett. (Coinedthe term chromatography)

General Chromatography Principle: The basic principle involved is, mixture of molecules on the surface of solid or liquid gets separated in the stationary phase(which is also called stable phase) from each other and moves with the help of mobile phase. Factors leading to separation generally includes adsorption (liquid-solid), partition(liquid-solid) and affinity, that is molecules separate based on their molecular weights. S- phase is solid or liquid while M- phase can be liquid or gas.

Various chromatography methods involved in enzyme purification:

  1. Ion exchange chromatography
  2. Affinity chromatography
  3. Size exclusion chromatography/gel permeation chromatography
  4. Immunoaffinity chromatography
  5. Hydrophobic interaction chromatography
  1. Ion exchange chromatography:
  2. In this method polar molecules or ions gets separated depending on their affinity to ion exchangers.
  3. Cationic exchangers: They are basically negatively charged and attracts positively charged cations. Due to ionisation of acidic group, they are charged negatively and hence also known as acid ion exchange materials.
  4. Anionic exchangers: also called basic ion exchange materials. They are charged positively and attracts negatively charged anions.

  Working Principle of ion exchange chromatography:

  • The technique is based on attractions between oppositely charged stationary phase, which is basically an ion and an analyte.
  • Ion exchangers are charged groups, linked covalently to surface matrix.(can be positive or negative).
  • Charged groups, when suspended in aqueous solution will be surrounded by oppositely charged ions.
  • This forms an ‘ion cloud’ . In this ion cloud exchange of ions occurs without altering the property and nature of the matrix.

 Instrumentation:

  • Pump- IC pump is present which helps in continuous supply and flow of eluent(carrier portion- portion carrying the molecule). In this the eluent used is liquid.
  • Injector: The instrument is equipped with an injector valve that injects or allows the liquid to pass through. Solid substances are first dissolved in solvent and than they are injected through the valve.(injecting range of liquid ranges from 0.1-100ml of volume).
  • Columns: material of the column depends on the application of use. It can be various types like, glass, steel, titanium and inert plastic such as PEEK. Column diameter ranges from 2mm-5cm. Guard column is placed inside separating column for ensuring the safety of the column and its usage for longer durations.
  • Suppressor: To reduce background conductivity of chemicals used for sample elution, suppressors are used. IC suppressors are employed to convert ionic eluent water.(enhances the sensitivity).
  • Detector: commonly used detector is electrical conductivity detector.

Data system: connected to a data system to obtain high throughput data.

Procedure of ion exchange chromatography:

  • Columns are used for packaging and packed with ion exchangers.
  • Commercially available ion exchangers are made up of Styrene and Di-vinyl chloride.
  • Based on charge of particle to be separated, ion exchangers are selected.
  • The column contains sample, ion exchanger and buffer.( Tris and acetate buffer are used widely).
  • Particles gets separated based on its affinity towards ion exchangers. Particles with higher affinity for ion exchangers, settles down at the bottom of the column along with buffer.
  • Spectroscopy methods are used for sample analysis.

Merit of the method: used for separating charged particles. In-organic ions can also be separated by using this method.

Demerit: Major drawback is, only charged molecules can be separated.

  • Affinity Chromatography:
  • The technique is based on affinity phenomenon, in which atoms are held intact in combination in a mixture by exerting an attracting force between the atoms.
  • Example can be enzyme and inhibitors.
  • Affinity based chromatography was first used and demonstrated Meir Wilcheck and Pedro Cuatrecasas.

Principle:

  • Substrate(ligand) molecules binds covalently on the support medium in stationary phase. The reactive molecules required for binding to the target are exposed.
  • Using chromatography column, the mixture is allowed to pass through. Substances binding to Immobilized substrate binding sites, will also bind to stationary phase, while the leftover mixture is eluted in the volume void of the column.
  • Target molecules which remain attached to the target can be eluted by altering pH, polarity or ionic strength of the solution.

Components of affinity chromatography:

  • Matrix: coupling of ligand to the target molecule takes place in the matrix. It is very important to have a proper matrix for affinity chromatography. The matrix should be chemically and physically inert, it should have a larger surface area for more adsorption of molecules, matrix should be insoluble in buffers and solvents. Polyacrylamide and agarose are the common materials used in matrix preparation.
  • Spacer Arm: Target molecule binds with the ligand with the help of spacer arm. Spacer arm facilitates this binding by avoiding steric hindrance.(rate of reaction is slow due to bulking of large molecules and atoms).

Ligand: molecule binding reversibly to the target molecule is ligand. Based on the nature of macromolecules isolated, ligand can be selected. For purification of enzymes, cofactor, substrate analogue or inhibitor is used as a ligand.

Procedure:

  • Column preparation: materials like cellulose, cephalose or agarose are used for column packaging. Ligand selection is based on the sample opted for affinity chromatography.
  • Sample loading: The loaded mixture of substances is poured into an elution column. Elution column allows the sample to run at controlled rate.
  • Elution: Target substance recovery is done using elution by changing pH, ionic strength and polarity conditions.
  • Used in most of the enzymatic assays for identifying binding sites of enzymes.

Merits: Specificity is very high in this method, target molecules in pure form can be obtained, the matrix used can be reused and gives higher yields.

Demerits: number of solvents required are more, if pH is not adjusted properly, proteins gets denatured, and one major drawback is eliminating non-specific adsorption.

  • Size Exclusion chromatography:
  • This technique can be used in particular for high molecular mass specie.
  • Porous material is S-phase and mobile phase is liquid.
  • Diameter of the pores of porous material used generally ranges from 50 to 3000 A.(A- angstrom).
  • Molecules which are smaller in size penetrates the membrane faster than larger molecules.
  • Size of solute molecule is used as a separation measure in this chromatography method.

Principle:

  • In this method, molecules are selected based on their molecular weights and size.
  • For molecules to be separated, porous glass granules and the selected liquid solvent are in equilibrium.

As smaller molecules move faster than larger molecules, larger molecules which are excluded from the column will pass through the interstitial spaces, where generally smaller molecules are distributed between inside and outside of the solvent sieve, which will than move at much slower rates.

Theory:

  • Volume of column in total is given as:

(Vt= Vg +Vi+ Vo), where Vg, is volume occupied by solid matrix, Vi is solvent volume and Vo is free volume, volume outside particles.

Components and procedure:

  • Dextran, agarose, polyacrylamide are the commonly used gels.
  • Column packaging: done by using silica glass granules or by  cross-linked organic gels such as dextran.
  • Detectors: detectors to be used are decided based on UV fluorescence and refractive index.
  • Size Exclusion chromatography can be implied in purification of enzymes and is extensively used in enzymatic assays.

Merits: larger components can be separated from smaller ones, shorter analysis time, no loss of the sample, gives narrow bands with good sensitivity.

Demerits: filtering in mobile phase is mandatory to avoid columns interfering with detectors and one major disadvantage is that it takes shorter time, the mount of peaks resolved are less, selectivity is also poor compared to other techniques.

  • Immunoaffinity Chromatography:
  • This method is used for separating antigen or antibody from heterogeneous mixture.
  • This method is used as combined method with LC(liquid chromatography) for binding of specific antibody or antigens.

Principle:

  • S-phase is antibody or antibody related agent.
  • The method is column based in which solution is allowed to flow through the column followed by elution.
  • Antigen or antibody is pre-functionalised in the column before the start of experiment.
  • Resin bound capture protein absorbs the target protein, while the leftover solution is eluted.
  • The fraction with target protein is also eluted and purified.

Immunoaffinity Chromatography Considerations:

  • Good column material is essential for the efficiency of the technique. The column should have:  Higher efficiency, mechanical stability, lower nonspecific binding.
  • Optimal size of pores. Smaller the pores, higher the surface area, but it won’t be accessible to larger proteins.
  • Larger pores have lower immobilization.

Antibody attachment:

  • Antibodies attach to column via covalent bonding.
  • Orientation of antibody and it’s attachment point are of important consideration in this type of method.
  • At FAB(fragment antigen binding) region antibody binding is not possible.(this site’s have to be unbound, for antigen to bind).
  • Antibody can be attached via carbohydrate residues, or direct attachment via amines or carboxyl’s.

Components and working:

  • Traditional columns in this method are made up of cellulose or agarose.
  • In high performance Immunoaffinity methods, columns are made up of silica and Azalactone beads.
  • Hydrophobic interactions chromatography: (HIC)
  • Molecules are separated based on hydrophobicity, that is lack of affinity towards water molecules.
  • Molecules elute by decreasing polarity of buffer.
  • If the molecule is more hydrophobic, the binding will be more stronger.

Principle:

  • Samples are loaded with high salt buffers containing hydrobhic and hydrophilic regions.
  • Solvation of sample solutes is reduced by salt buffer.
  • Due to decrease in solvation, hydrophobic regions gets adsorbed by the media.
  • Less salt is required for binding of the molecule is more hydrophobic.
  • During elution, decreasing salt gradient is used.
  • Sample elution can be also done by detergents or modifiers added in the elution buffer.

General considerations in HIC:

  • Ligand: Behaviour of enzymes or proteins can be determined by Immobilized ligand used. For example- straight chain alkyl ligands exhibits hydrophobic nature.
  • Degree of substitution: degree of ligand substitution is directly proportional to binding capacity of enzyme or protein.
  • Temperature: correlation between hydrophobic interactions and temperature is affects solubility and structure of enzyme or protein.
  • pH- mobile phase used in HIC, usually have pH in the range of 5-7. Effect of pH differs from protein-protein.
  • Salt concentrations: salt concentrations shows higher ligand-protein binding but higher concentrations can lead to protein precipitation.

Procedure of HIC:

  • Media is made of alkyl or aryl ligands.
  • Space between matrix is filled by moderate salt buffers.
  • Non-bound proteins can be eliminated by washing the column.
  • Lower the salt concentration, during elution.
  • Ethanol(70%) can be used to remove unbound proteins.

Merits: high ionic strength samples can be used. Large volume of samples can be loaded easily.

Demerits: major drawback is requirement of non-volatile mobile phase.

Thus, these chromatography techniques are essential for purification of enzymes.

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